proteinase k  (Roche)

 
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    Proteinase K solution
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    PKS8773949
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    Structured Review

    Roche proteinase k
    PrP CWD  amplification in raw water samples from non-CWD-endemic areas. All samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. Lane 2 shows amplified NBH control. sPMCA failed to amplify any PrP CWD  from five replicate

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    Images

    1) Product Images from "Detection of protease-resistant cervid prion protein in water from a CWD-endemic area"

    Article Title: Detection of protease-resistant cervid prion protein in water from a CWD-endemic area

    Journal:

    doi:

    PrP CWD  amplification in raw water samples from non-CWD-endemic areas. All samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. Lane 2 shows amplified NBH control. sPMCA failed to amplify any PrP CWD  from five replicate
    Figure Legend Snippet: PrP CWD amplification in raw water samples from non-CWD-endemic areas. All samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. Lane 2 shows amplified NBH control. sPMCA failed to amplify any PrP CWD from five replicate

    Techniques Used: Amplification

    Effects of flocculation and alum on PrP CWD  amplification. All samples were digested with Proteinase K except lane 1. (A) PrP CWD  precipitates with flocculant water sample. Lanes 1 and 2 shows amplified NBH control. Lanes 3–6 show amplified samples
    Figure Legend Snippet: Effects of flocculation and alum on PrP CWD amplification. All samples were digested with Proteinase K except lane 1. (A) PrP CWD precipitates with flocculant water sample. Lanes 1 and 2 shows amplified NBH control. Lanes 3–6 show amplified samples

    Techniques Used: Flocculation, Amplification

    PrP CWD  detection limit in water. (A) All samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. Lanes 2 through 12 show amplified samples at the indicated starting dilution of CWD-positive brain into water. Lanes 13 and
    Figure Legend Snippet: PrP CWD detection limit in water. (A) All samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. Lanes 2 through 12 show amplified samples at the indicated starting dilution of CWD-positive brain into water. Lanes 13 and

    Techniques Used: Amplification

    PrP CWD  detected in raw water samples collected at a time of increased snow-melt runoff. All samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. Lane 2 shows amplified NBH control. Lanes 3–8 show Horsetooth reservoir
    Figure Legend Snippet: PrP CWD detected in raw water samples collected at a time of increased snow-melt runoff. All samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. Lane 2 shows amplified NBH control. Lanes 3–8 show Horsetooth reservoir

    Techniques Used: Amplification

    Abrogation of PrP CWD  amplification from 5-22-07 raw PR water samples by PMCA using murine PrP C  substrate or Proteinase K pre-treatment. All western blot samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. Lane 2 shows
    Figure Legend Snippet: Abrogation of PrP CWD amplification from 5-22-07 raw PR water samples by PMCA using murine PrP C substrate or Proteinase K pre-treatment. All western blot samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. Lane 2 shows

    Techniques Used: Amplification, Western Blot

    Normal brain homogenate negative controls. All samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. NBH, Normal brain homogenate control. +, 1:100,000 positive amplification control. Molecular weight markers in kilodaltons
    Figure Legend Snippet: Normal brain homogenate negative controls. All samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. NBH, Normal brain homogenate control. +, 1:100,000 positive amplification control. Molecular weight markers in kilodaltons

    Techniques Used: Amplification, Molecular Weight

    2) Product Images from "Characterization of four new monoclonal antibodies against the distal N-terminal region of PrPc"

    Article Title: Characterization of four new monoclonal antibodies against the distal N-terminal region of PrPc

    Journal: PeerJ

    doi: 10.7717/peerj.811

    N-terminal mAbs can inhibit prion replication. RML infected GT1 cells were treated for 6 days with increasing concentrations (0, 1, 2.5, 5 and 7.5 µg/mL) of EB8, DE10, DC2 and EF2 mAbs, refreshing medium the third day. Cell lysates were digested with proteinase K (PK + lanes) and PrP Sc  levels checked by Western blot using Fab D18 for detection. As positive control (PC), cell lysates from uninfected GT1 cells were also digested. About 25 µg of total proteins were loaded as control (PK—lanes). DE10, DC2 and EF2 mAbs promoted a complete clearance of prions starting from the lowest concentration tested whilst cells treated with EB8 showed a residual signal of PrP Sc  even at the highest concentration of antibody. Images are representative of three independent experiments.
    Figure Legend Snippet: N-terminal mAbs can inhibit prion replication. RML infected GT1 cells were treated for 6 days with increasing concentrations (0, 1, 2.5, 5 and 7.5 µg/mL) of EB8, DE10, DC2 and EF2 mAbs, refreshing medium the third day. Cell lysates were digested with proteinase K (PK + lanes) and PrP Sc levels checked by Western blot using Fab D18 for detection. As positive control (PC), cell lysates from uninfected GT1 cells were also digested. About 25 µg of total proteins were loaded as control (PK—lanes). DE10, DC2 and EF2 mAbs promoted a complete clearance of prions starting from the lowest concentration tested whilst cells treated with EB8 showed a residual signal of PrP Sc even at the highest concentration of antibody. Images are representative of three independent experiments.

    Techniques Used: Infection, Western Blot, Positive Control, Concentration Assay

    Time-course analysis of mAb-induced prion clearance. ScGT1 cells were incubated for 1 week with 5 µg/mL of mAbs. Untreated cells were used as negative control (NC). After the initial treatment, cells were split and cultured in absence of mAbs for 1 month. Cell lysates were digested with proteinase K (PK + lanes) and PrP Sc  was probed by Western blot using Fab D18. PrP Sc  levels were analyzed after one (1 w), two (2 w), three (3 w) and four (4 w) weeks after the treatment to evaluate the stability of clearance during time. Prions were not detectable in treated cells one month after the mAbs incubation. Just a slight signal from PrP Sc  was found in EB8 treated ScGT1 cells. Images are representative of three independent experiments. Lanes were run on the same gel but were non-contiguous (white lines).
    Figure Legend Snippet: Time-course analysis of mAb-induced prion clearance. ScGT1 cells were incubated for 1 week with 5 µg/mL of mAbs. Untreated cells were used as negative control (NC). After the initial treatment, cells were split and cultured in absence of mAbs for 1 month. Cell lysates were digested with proteinase K (PK + lanes) and PrP Sc was probed by Western blot using Fab D18. PrP Sc levels were analyzed after one (1 w), two (2 w), three (3 w) and four (4 w) weeks after the treatment to evaluate the stability of clearance during time. Prions were not detectable in treated cells one month after the mAbs incubation. Just a slight signal from PrP Sc was found in EB8 treated ScGT1 cells. Images are representative of three independent experiments. Lanes were run on the same gel but were non-contiguous (white lines).

    Techniques Used: Incubation, Negative Control, Cell Culture, Western Blot

    3) Product Images from "Preclinical deposition of pathological prion protein PrPSc in muscles of hamsters orally exposed to scrapie"

    Article Title: Preclinical deposition of pathological prion protein PrPSc in muscles of hamsters orally exposed to scrapie

    Journal:

    doi: 10.1172/JCI200421083

    Time course of PrPSc  deposition in muscle tissue. Western blot detection of PrP27-30 extracted from different muscles and sciatic nerve of hamsters orally challenged with 263K scrapie and sacrificed at the following time points after infection. ( A ) At 100 days after infection, ( B – D ) 130 days after infection, ( E ) onset of clinical signs for scrapie (139–149 days after infection), and ( F ) at the terminal stage of disease (161–173 days after infection). Lanes with test samples: M1, M. biceps femoris (hindlimb); M2, M. tibialis cranialis (hindlimb); M3, M. triceps brachii (forelimb); M4, M. extensor carpi radialis (forelimb); M5, M. trapezius (shoulder); M6, M. masseter (head); M7, M. psoas major (back); T, tongue; H, heart; SN, sciatic nerve. Lanes with control samples: 1, proteinase K–digested brain homogenate from terminally ill scrapie hamsters containing 1 ∞ 10–7  g ( B  and  F ) or 5 ∞ 10–7  g ( A ,  C – E ) brain tissue; 2, skeletal muscle from an uninfected hamster spiked before extraction with 5 ∞ 10–6  g ( A ,  B , and  F ), 1 ∞ 10–5  g ( C  and  D ), or 2 ∞ 10–5  g ( E ) brain homogenate from terminally ill scrapie hamsters; 3, skeletal muscle from a mock-challenged hamster sacrificed at 173 days after infection ( F ). For each stage of incubation representative results are shown. ( G ) Time scale displaying the mean incubation period and the preclinical and clinical phases of incubation of hamsters orally infected with 263K scrapie. Small vertical arrows indicate time points at which animals were screened for PrPSc  in muscles. dai, days after infection.
    Figure Legend Snippet: Time course of PrPSc deposition in muscle tissue. Western blot detection of PrP27-30 extracted from different muscles and sciatic nerve of hamsters orally challenged with 263K scrapie and sacrificed at the following time points after infection. ( A ) At 100 days after infection, ( B – D ) 130 days after infection, ( E ) onset of clinical signs for scrapie (139–149 days after infection), and ( F ) at the terminal stage of disease (161–173 days after infection). Lanes with test samples: M1, M. biceps femoris (hindlimb); M2, M. tibialis cranialis (hindlimb); M3, M. triceps brachii (forelimb); M4, M. extensor carpi radialis (forelimb); M5, M. trapezius (shoulder); M6, M. masseter (head); M7, M. psoas major (back); T, tongue; H, heart; SN, sciatic nerve. Lanes with control samples: 1, proteinase K–digested brain homogenate from terminally ill scrapie hamsters containing 1 ∞ 10–7 g ( B and F ) or 5 ∞ 10–7 g ( A , C – E ) brain tissue; 2, skeletal muscle from an uninfected hamster spiked before extraction with 5 ∞ 10–6 g ( A , B , and F ), 1 ∞ 10–5 g ( C and D ), or 2 ∞ 10–5 g ( E ) brain homogenate from terminally ill scrapie hamsters; 3, skeletal muscle from a mock-challenged hamster sacrificed at 173 days after infection ( F ). For each stage of incubation representative results are shown. ( G ) Time scale displaying the mean incubation period and the preclinical and clinical phases of incubation of hamsters orally infected with 263K scrapie. Small vertical arrows indicate time points at which animals were screened for PrPSc in muscles. dai, days after infection.

    Techniques Used: Western Blot, Infection, Incubation

    4) Product Images from "Leishmania mortality in sand fly blood meal is not species-specific and does not result from direct effect of proteinases"

    Article Title: Leishmania mortality in sand fly blood meal is not species-specific and does not result from direct effect of proteinases

    Journal: Parasites & Vectors

    doi: 10.1186/s13071-018-2613-2

    Promastigotes of  L. donovani  incubated with sand fly midguts dissected at different times post-blood meal and various controls.  Leishmania donovani  promastigotes (24 h after the releasing from macrophages) were incubated in a microtiter plate for 2 h with:  a  midguts of  P. argentipes ,  P. orientalis ,  P. papatasi  and  S. schwetzi  dissected at 6, 24, 32, 48 and 72 h post-blood meal; or  b  with saline, proteinase K (PK), rabbit blood, red cells of rabbit blood, human haemoglobin, blood + proteinase K, red cells + proteinase K and human haemoglobin + proteinase K. As a positive control, parasites killed by 1% formaldehyde and permeabilised by 0.5% Triton X-100 were used
    Figure Legend Snippet: Promastigotes of L. donovani incubated with sand fly midguts dissected at different times post-blood meal and various controls. Leishmania donovani promastigotes (24 h after the releasing from macrophages) were incubated in a microtiter plate for 2 h with: a midguts of P. argentipes , P. orientalis , P. papatasi and S. schwetzi dissected at 6, 24, 32, 48 and 72 h post-blood meal; or b with saline, proteinase K (PK), rabbit blood, red cells of rabbit blood, human haemoglobin, blood + proteinase K, red cells + proteinase K and human haemoglobin + proteinase K. As a positive control, parasites killed by 1% formaldehyde and permeabilised by 0.5% Triton X-100 were used

    Techniques Used: Incubation, Positive Control

    5) Product Images from "The Amino-Terminal Part of the Needle-Tip Translocator LcrV of Yersinia pseudotuberculosis Is Required for Early Targeting of YopH and In vivo Virulence"

    Article Title: The Amino-Terminal Part of the Needle-Tip Translocator LcrV of Yersinia pseudotuberculosis Is Required for Early Targeting of YopH and In vivo Virulence

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2016.00175

    Translocation levels are up-regulated even in the absence of pore formation . Translocated YopH and YopE detected within HeLa cells after 1 h of infection (Δ yopK , Δ K/V + 1 , Δ K/V–1  and Δ K/ Δ V , Δ yopE  and Δ yopH ) or after 2 h (Wt, V+1, V−1, Δ lcrV ). Extracellular proteins were removed by Proteinase K treatment after which the cells were lysed with 1% Digitonin. One sample (Wt–Digi) was left unlysed as a negative control. Detection of tubulin served as a loading control. The experiment was repeated at least three times and a representative experiment is shown. The signal intensity of the bands was quantified using the Multi Gauge-Image software (Fujifilm). The values for YopE and YopH were normalized to the loading control and the ratio ± SEM of translocated Yops relative to the Δ yopK  mutant, is shown below each Western blot. The results were analyzed using the paired Student's  t -test with the significance set at  p  ≤ 0.01 ** . NS, No significant difference.
    Figure Legend Snippet: Translocation levels are up-regulated even in the absence of pore formation . Translocated YopH and YopE detected within HeLa cells after 1 h of infection (Δ yopK , Δ K/V + 1 , Δ K/V–1 and Δ K/ Δ V , Δ yopE and Δ yopH ) or after 2 h (Wt, V+1, V−1, Δ lcrV ). Extracellular proteins were removed by Proteinase K treatment after which the cells were lysed with 1% Digitonin. One sample (Wt–Digi) was left unlysed as a negative control. Detection of tubulin served as a loading control. The experiment was repeated at least three times and a representative experiment is shown. The signal intensity of the bands was quantified using the Multi Gauge-Image software (Fujifilm). The values for YopE and YopH were normalized to the loading control and the ratio ± SEM of translocated Yops relative to the Δ yopK mutant, is shown below each Western blot. The results were analyzed using the paired Student's t -test with the significance set at p ≤ 0.01 ** . NS, No significant difference.

    Techniques Used: Translocation Assay, Infection, Negative Control, Software, Mutagenesis, Western Blot

    6) Product Images from "Dissociation of Infectivity from Seeding Ability in Prions with Alternate Docking Mechanism"

    Article Title: Dissociation of Infectivity from Seeding Ability in Prions with Alternate Docking Mechanism

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002128

    Effect of accessory cofactors on the propagation of ΔPBD PrP Sc  molecules. In vitro -generated ΔC-PBD, ΔN-PBD, and ΔΔ-PBD PrP Sc  molecules were propagated by sPMCA with autologous PrP C  prepared from Chinese hamster ovary (CHO) cells. One set of reactions was supplemented with  Prnp 0/0  mouse brain homogenate (+cofactor), while a second set received only buffer (−cofactor). One sample of each reaction was not subjected to protease digestion (−PK), while all others were digested with 25 µg/mL proteinase K. –PK reactions for ΔC-PBD and ΔΔ-PBD were loaded with ¼ volume. PrP was detected by immunoblot (anti-PrP 27/33).
    Figure Legend Snippet: Effect of accessory cofactors on the propagation of ΔPBD PrP Sc molecules. In vitro -generated ΔC-PBD, ΔN-PBD, and ΔΔ-PBD PrP Sc molecules were propagated by sPMCA with autologous PrP C prepared from Chinese hamster ovary (CHO) cells. One set of reactions was supplemented with Prnp 0/0 mouse brain homogenate (+cofactor), while a second set received only buffer (−cofactor). One sample of each reaction was not subjected to protease digestion (−PK), while all others were digested with 25 µg/mL proteinase K. –PK reactions for ΔC-PBD and ΔΔ-PBD were loaded with ¼ volume. PrP was detected by immunoblot (anti-PrP 27/33).

    Techniques Used: In Vitro, Generated

    Biochemical and neuropathological analysis of mice inoculated with  in vitro -generated PrP Sc  molecules. Brains were dissected from wild-type mice showing terminal scrapie signs (PrP Sc  and ΔC-PBD PrP Sc  inocula) or similarly aged mice not displaying scrapie signs (mock-propagated, ΔN-PBD PrP Sc , and ΔΔ-PBD PrP Sc  inocula). ( A ) Equivalent amounts of 10% brain homogenate were treated with buffer (−PK) or 25 µg/mL proteinase K (+PK to show PrP Sc ) and detected by anti-PrP (6D11) immunoblot. A greater exposure of the same immunoblot is displayed below, to illustrate samples containing low amounts of PrP Sc . ( B ) Neuropathology of cerebellum and hippocampus. Brain sections were stained with hematoxylin and eosin (H  E). The black bar denotes 100 µm.
    Figure Legend Snippet: Biochemical and neuropathological analysis of mice inoculated with in vitro -generated PrP Sc molecules. Brains were dissected from wild-type mice showing terminal scrapie signs (PrP Sc and ΔC-PBD PrP Sc inocula) or similarly aged mice not displaying scrapie signs (mock-propagated, ΔN-PBD PrP Sc , and ΔΔ-PBD PrP Sc inocula). ( A ) Equivalent amounts of 10% brain homogenate were treated with buffer (−PK) or 25 µg/mL proteinase K (+PK to show PrP Sc ) and detected by anti-PrP (6D11) immunoblot. A greater exposure of the same immunoblot is displayed below, to illustrate samples containing low amounts of PrP Sc . ( B ) Neuropathology of cerebellum and hippocampus. Brain sections were stained with hematoxylin and eosin (H E). The black bar denotes 100 µm.

    Techniques Used: Mouse Assay, In Vitro, Generated, Staining

    Interaction of ΔC-PBD PrP Sc  with mutant PrP molecules. ( A ) Binding of ΔC-PBD PrP Sc  to PrP. ΔC-PBD PrP Sc  was incubated with wild-type or polybasic mutant (ΔC-PBD, ΔN-PBD) myc-tagged PrP. Bound PrP Sc  was captured with 9E10 anti-myc antibody on magnetic protein A Dynabeads, and detected by 25 µg/mL proteinase K digestion and anti-PrP (27/33) immunoblot. ( B ) Propagation of ΔC-PBD PrP Sc . ΔC-PBD PrP Sc  was propagated by sPMCA with polybasic deletion mutant PrP C  prepared from Chinese hamster ovary (CHO) cells. Reactions were supplemented with  Prnp 0/0  mouse brain homogenate. One sample of each reaction was not subjected to protease digestion (−PK), loading ¼ of volume. All others were subjected to limited proteolysis by 25 µg/mL proteinase K digestion. PrP was detected by immunoblot (anti-PrP 27/33).
    Figure Legend Snippet: Interaction of ΔC-PBD PrP Sc with mutant PrP molecules. ( A ) Binding of ΔC-PBD PrP Sc to PrP. ΔC-PBD PrP Sc was incubated with wild-type or polybasic mutant (ΔC-PBD, ΔN-PBD) myc-tagged PrP. Bound PrP Sc was captured with 9E10 anti-myc antibody on magnetic protein A Dynabeads, and detected by 25 µg/mL proteinase K digestion and anti-PrP (27/33) immunoblot. ( B ) Propagation of ΔC-PBD PrP Sc . ΔC-PBD PrP Sc was propagated by sPMCA with polybasic deletion mutant PrP C prepared from Chinese hamster ovary (CHO) cells. Reactions were supplemented with Prnp 0/0 mouse brain homogenate. One sample of each reaction was not subjected to protease digestion (−PK), loading ¼ of volume. All others were subjected to limited proteolysis by 25 µg/mL proteinase K digestion. PrP was detected by immunoblot (anti-PrP 27/33).

    Techniques Used: Mutagenesis, Binding Assay, Incubation

    ΔPBD PrP Sc  molecules seeding wild-type brain homogenate sPMCA reactions. In vitro -generated ΔC-PBD, ΔN-PBD, and ΔΔ-PBD PrP Sc  molecules were propagated by sPMCA for three rounds with wild-type mouse brain homogenate, containing wild-type PrP C  substrate. Control reactions seeded with wild-type native RML prions and unseeded reactions were also tested. One sample of each reaction was not subjected to protease digestion (−PK), while all others were digested with 25 µg/mL proteinase K. PrP was detected by immunoblot (anti-PrP 6D11).
    Figure Legend Snippet: ΔPBD PrP Sc molecules seeding wild-type brain homogenate sPMCA reactions. In vitro -generated ΔC-PBD, ΔN-PBD, and ΔΔ-PBD PrP Sc molecules were propagated by sPMCA for three rounds with wild-type mouse brain homogenate, containing wild-type PrP C substrate. Control reactions seeded with wild-type native RML prions and unseeded reactions were also tested. One sample of each reaction was not subjected to protease digestion (−PK), while all others were digested with 25 µg/mL proteinase K. PrP was detected by immunoblot (anti-PrP 6D11).

    Techniques Used: In Vitro, Generated

    Interaction of wild-type PrP Sc  with mutant PrP molecules. ( A ) Binding of PrP Sc  to PrP. RML scrapie-infected mouse brain homogenate was incubated with myc-tagged PrP of wild-type sequence or lacking the central (ΔC-PBD: Δ100–109) or N-terminal (ΔN-PBD: Δ23–28) polybasic domain. Bound PrP Sc  was captured with 9E10 anti-myc antibody on magnetic protein A Dynabeads, and detected by 25 µg/mL proteinase K digestion and anti-PrP (6D11) immunoblot. ( B ) Propagation of PrP Sc . RML scrapie-infected mouse brain homogenate was propagated by serial protein misfolding cyclic amplification (sPMCA) for five rounds with wild-type or polybasic deletion mutant PrP C  prepared from Chinese hamster ovary (CHO) cells. Reactions were supplemented with  Prnp 0/0  mouse brain homogenate. In addition to scrapie-seeded reactions, an unseeded reaction was performed with ΔC-PBD PrP substrate. One sample of each reaction was not subjected to protease digestion (−PK). All others were subjected to limited proteolysis with 25 µg/mL proteinase K. PrP was detected by immunoblot (anti-PrP 27/33).
    Figure Legend Snippet: Interaction of wild-type PrP Sc with mutant PrP molecules. ( A ) Binding of PrP Sc to PrP. RML scrapie-infected mouse brain homogenate was incubated with myc-tagged PrP of wild-type sequence or lacking the central (ΔC-PBD: Δ100–109) or N-terminal (ΔN-PBD: Δ23–28) polybasic domain. Bound PrP Sc was captured with 9E10 anti-myc antibody on magnetic protein A Dynabeads, and detected by 25 µg/mL proteinase K digestion and anti-PrP (6D11) immunoblot. ( B ) Propagation of PrP Sc . RML scrapie-infected mouse brain homogenate was propagated by serial protein misfolding cyclic amplification (sPMCA) for five rounds with wild-type or polybasic deletion mutant PrP C prepared from Chinese hamster ovary (CHO) cells. Reactions were supplemented with Prnp 0/0 mouse brain homogenate. In addition to scrapie-seeded reactions, an unseeded reaction was performed with ΔC-PBD PrP substrate. One sample of each reaction was not subjected to protease digestion (−PK). All others were subjected to limited proteolysis with 25 µg/mL proteinase K. PrP was detected by immunoblot (anti-PrP 27/33).

    Techniques Used: Mutagenesis, Binding Assay, Infection, Incubation, Sequencing, Protein Misfolding Cyclic Amplification

    7) Product Images from "Transgenic Rabbits Expressing Ovine PrP Are Susceptible to ScrapieTransgenic Mouse Bioassay: Evidence That Rabbits Are Susceptible to a Variety of Prion Isolates"

    Article Title: Transgenic Rabbits Expressing Ovine PrP Are Susceptible to ScrapieTransgenic Mouse Bioassay: Evidence That Rabbits Are Susceptible to a Variety of Prion Isolates

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1005077

    Brain PrP Sc  in ovine PrP transgenic rabbit infected with LA21K fast scrapie prions. (a) Western blot analyses of the brain from wild-type and tgOv rabbits mock-infected or inoculated with LA21K  fast  prions for the presence of proteinase K (PK)-resistant PrP Sc . The equivalent of 2 mg brain tissue were loaded in lane 1–6, 3 mg in lanes 7 and 8. (b) Electrophoretic pattern and (c) glycoform ratios (plotted as means ± SEM(c)) of PrP res  in the brains and spleens of tgOv rabbit and tg338 mice infected with LA21K fast prions. The same amount of brain (0.5 mg) and spleen (3 mg) tissue equivalent was loaded on the gel.
    Figure Legend Snippet: Brain PrP Sc in ovine PrP transgenic rabbit infected with LA21K fast scrapie prions. (a) Western blot analyses of the brain from wild-type and tgOv rabbits mock-infected or inoculated with LA21K fast prions for the presence of proteinase K (PK)-resistant PrP Sc . The equivalent of 2 mg brain tissue were loaded in lane 1–6, 3 mg in lanes 7 and 8. (b) Electrophoretic pattern and (c) glycoform ratios (plotted as means ± SEM(c)) of PrP res in the brains and spleens of tgOv rabbit and tg338 mice infected with LA21K fast prions. The same amount of brain (0.5 mg) and spleen (3 mg) tissue equivalent was loaded on the gel.

    Techniques Used: Transgenic Assay, Infection, Western Blot, Mouse Assay

    8) Product Images from "Identification of an activation site in Bak and mitochondrial Bax triggered by antibodies"

    Article Title: Identification of an activation site in Bak and mitochondrial Bax triggered by antibodies

    Journal: Nature Communications

    doi: 10.1038/ncomms11734

    The 7D10 antibody triggers mitochondrial outer membrane permeabilization by binding to the α1–α2 loop of human Bak. ( a ) The 7D10 antibody induces cytochrome  c  release. Membrane fractions from  Bak −/− Bax −/−  MEFs, those cells expressing human Bak (hBak), or  Bax −/−  MEFs, were incubated with tBid or with the 7D10 or 8F8 antibodies. Supernatant (Sup) and pellet (Mito) fractions were assessed for cytochrome  c  release. ( b ) 7D10 triggers Bak conformational change as indicated by susceptibility to proteinase K. Incubations from  a  were treated with proteinase K and immunoblotted for Bak. Note that 7D10 binding at the loop masks a cleavage site (lane 4,  Supplementary Fig. 1a ), and that uncleaved Bak and light chain co-migrate. ( c ) 7D10 triggers Bak oligomerization. Incubations from  a  were treated with oxidant (CuPhe) to induce disulfide bond formation and immunoblotted for Bak. M, monomer; M x,  intramolecular linked monomers; D, intermolecular linked dimers. ( d ) The 7D10 trigger site in Bak is distinct from the canonical BH3-only trigger site. Cartoon representation of BakΔN19ΔC25 (2IMT, white) highlighting the α1–α2 loop (blue), and α3 and α4 of the hydrophobic groove (green). ( e ) Mutation of Bak G51 or P55 inhibits binding by 7D10. Membrane fractions from  Bak −/− Bax −/−  MEFs expressing the indicated hBak variants were incubated with or without tBid followed by immunoprecipitation with 7D10 and immunoblotting for Bak. IP, immunoprecipitated; UB, unbound; #, light chain. ( f ) Mutation of Bak G51 or P55 prevent Bak activation and cytochrome  c  release by 7D10. Membrane fractions from  e  were incubated with tBid or 7D10 and assessed for cytochrome  c  release. ( g ) Substitutions in mouse Bak to generate the 7D10 epitope. ( h ) The  51 GVAAPAD 57  sequence allows binding of 7D10 to mouse Bak. Membrane fractions from  Bax −/−  MEFs (mBak cells), or  Bak −/− Bax −/−  MEFs expressing hBak or the indicated mBak variants, were incubated with or without tBid followed by immunoprecipitation with 7D10 and immunoblotting for Bak. ( i ) The  51 GVAAPAD 57  sequence allows 7D10 to activate mouse Bak and release cytochrome  c . Membrane fractions from  h  were incubated with tBid or 7D10 and assessed for cytochrome  c  release. In  a – c , e , f , h  and  i  data are representative of three independent experiments.
    Figure Legend Snippet: The 7D10 antibody triggers mitochondrial outer membrane permeabilization by binding to the α1–α2 loop of human Bak. ( a ) The 7D10 antibody induces cytochrome c release. Membrane fractions from Bak −/− Bax −/− MEFs, those cells expressing human Bak (hBak), or Bax −/− MEFs, were incubated with tBid or with the 7D10 or 8F8 antibodies. Supernatant (Sup) and pellet (Mito) fractions were assessed for cytochrome c release. ( b ) 7D10 triggers Bak conformational change as indicated by susceptibility to proteinase K. Incubations from a were treated with proteinase K and immunoblotted for Bak. Note that 7D10 binding at the loop masks a cleavage site (lane 4, Supplementary Fig. 1a ), and that uncleaved Bak and light chain co-migrate. ( c ) 7D10 triggers Bak oligomerization. Incubations from a were treated with oxidant (CuPhe) to induce disulfide bond formation and immunoblotted for Bak. M, monomer; M x, intramolecular linked monomers; D, intermolecular linked dimers. ( d ) The 7D10 trigger site in Bak is distinct from the canonical BH3-only trigger site. Cartoon representation of BakΔN19ΔC25 (2IMT, white) highlighting the α1–α2 loop (blue), and α3 and α4 of the hydrophobic groove (green). ( e ) Mutation of Bak G51 or P55 inhibits binding by 7D10. Membrane fractions from Bak −/− Bax −/− MEFs expressing the indicated hBak variants were incubated with or without tBid followed by immunoprecipitation with 7D10 and immunoblotting for Bak. IP, immunoprecipitated; UB, unbound; #, light chain. ( f ) Mutation of Bak G51 or P55 prevent Bak activation and cytochrome c release by 7D10. Membrane fractions from e were incubated with tBid or 7D10 and assessed for cytochrome c release. ( g ) Substitutions in mouse Bak to generate the 7D10 epitope. ( h ) The 51 GVAAPAD 57 sequence allows binding of 7D10 to mouse Bak. Membrane fractions from Bax −/− MEFs (mBak cells), or Bak −/− Bax −/− MEFs expressing hBak or the indicated mBak variants, were incubated with or without tBid followed by immunoprecipitation with 7D10 and immunoblotting for Bak. ( i ) The 51 GVAAPAD 57 sequence allows 7D10 to activate mouse Bak and release cytochrome c . Membrane fractions from h were incubated with tBid or 7D10 and assessed for cytochrome c release. In a – c , e , f , h and i data are representative of three independent experiments.

    Techniques Used: Binding Assay, Expressing, Incubation, Mutagenesis, Immunoprecipitation, Activation Assay, Sequencing

    9) Product Images from "Enrichment of extracellular vesicles from human synovial fluid using size exclusion chromatography"

    Article Title: Enrichment of extracellular vesicles from human synovial fluid using size exclusion chromatography

    Journal: Journal of Extracellular Vesicles

    doi: 10.1080/20013078.2018.1490145

    Proteomic analysis of EVs isolated from synovial fluid of a rheumatoid arthritis patient. (a) Venn diagram comparing proteins identified by mass spectrometry analysis of SEC EVs in the presence or absence of proteinase K. About 652 unique proteins were identified without proteinase K treatment, and this number reduced to 270 following the addition of proteinase K. (b–d) GO analysis of the proteins identified from mass spectrometry analysis of proteinase K treated sample including cellular function and biological processes.
    Figure Legend Snippet: Proteomic analysis of EVs isolated from synovial fluid of a rheumatoid arthritis patient. (a) Venn diagram comparing proteins identified by mass spectrometry analysis of SEC EVs in the presence or absence of proteinase K. About 652 unique proteins were identified without proteinase K treatment, and this number reduced to 270 following the addition of proteinase K. (b–d) GO analysis of the proteins identified from mass spectrometry analysis of proteinase K treated sample including cellular function and biological processes.

    Techniques Used: Isolation, Mass Spectrometry, Size-exclusion Chromatography, Cell Function Assay

    10) Product Images from "A Novel Mitosomal β-Barrel Outer Membrane Protein in Entamoeba"

    Article Title: A Novel Mitosomal β-Barrel Outer Membrane Protein in Entamoeba

    Journal: Scientific Reports

    doi: 10.1038/srep08545

    Mitosomal membrane localization of MBOMP30. (a) Na 2 CO 3  fractionation. Homogenates from amoebae expressing HA-MBOMP30 and Tom40-HA were fractionated. The organelle fraction was treated with Na 2 CO 3  and NaCl to lyse the organelle and liberate loosely bound proteins. The resulting fractions were separated on SDS-PAGE followed by immunoblotting. Parts of the full-length immunoblot for the anti-HA (first two rows) and anti-Cpn60 (last row) antibody reactions are shown respectively. Tom40-HA and Cpn60 serve as control for mitosomal BOMP and soluble mitosomal protein, respectively. The original blots are shown in  Supplementary Fig. S4a–b . (b) Proteinase K protection assay. Cropped immunoblots of proteinase K-treated (+) and untreated (−) organelle fractions are shown on the left panel, with the corresponding ratio of protein digestion on the right panel (See full-length immunoblots on  Supplementary Fig. S4c ) (c) Immunoelectron microscopy. Immunodecoration of mitosomes of HA-MBOMP30, HA-MBOMP30 Δ275–282 , and control trophozoites with anti-HA (15 nm gold particles) and anti-Cpn60 antibodies (5 nm gold particles) showed electron-dense organelles surrounded by double membranes [scale bar, 200 nm].
    Figure Legend Snippet: Mitosomal membrane localization of MBOMP30. (a) Na 2 CO 3 fractionation. Homogenates from amoebae expressing HA-MBOMP30 and Tom40-HA were fractionated. The organelle fraction was treated with Na 2 CO 3 and NaCl to lyse the organelle and liberate loosely bound proteins. The resulting fractions were separated on SDS-PAGE followed by immunoblotting. Parts of the full-length immunoblot for the anti-HA (first two rows) and anti-Cpn60 (last row) antibody reactions are shown respectively. Tom40-HA and Cpn60 serve as control for mitosomal BOMP and soluble mitosomal protein, respectively. The original blots are shown in Supplementary Fig. S4a–b . (b) Proteinase K protection assay. Cropped immunoblots of proteinase K-treated (+) and untreated (−) organelle fractions are shown on the left panel, with the corresponding ratio of protein digestion on the right panel (See full-length immunoblots on Supplementary Fig. S4c ) (c) Immunoelectron microscopy. Immunodecoration of mitosomes of HA-MBOMP30, HA-MBOMP30 Δ275–282 , and control trophozoites with anti-HA (15 nm gold particles) and anti-Cpn60 antibodies (5 nm gold particles) showed electron-dense organelles surrounded by double membranes [scale bar, 200 nm].

    Techniques Used: Fractionation, Expressing, Hemagglutination Assay, SDS Page, Western Blot, Immuno-Electron Microscopy

    11) Product Images from "Improved delivery of the OVA-CD4 peptide to T helper cells by polymeric surface display on Salmonella"

    Article Title: Improved delivery of the OVA-CD4 peptide to T helper cells by polymeric surface display on Salmonella

    Journal: Microbial Cell Factories

    doi: 10.1186/1475-2859-13-80

    Expression of OVA-CD4pep-MisL fusion proteins determined by western blot analysis using an anti-OVA-CD4 peptide polyclonal antibody (A) Ovalbumin (lane 1), strain CS4551 with pnirBLTBsp-MisL (lanes 2 and 3), pZS1202 which expresses the (OVA-CD4)-MisL fusion protein from the  nirB  promoter (lanes 4 and 5), or pZS1204 expresses the (OVA-CD4)-MisL fusion protein from the  nirB  and  spiC  promoters in tandem (lanes 6 and 7); (B) Bovine serum albumin (lane 1), ovalbumin (lane 2), untreated (lane 3) or proteinase K-treated (lane 4) outer membrane proteins of  Salmonella  strain SL7207 pZ1204.
    Figure Legend Snippet: Expression of OVA-CD4pep-MisL fusion proteins determined by western blot analysis using an anti-OVA-CD4 peptide polyclonal antibody (A) Ovalbumin (lane 1), strain CS4551 with pnirBLTBsp-MisL (lanes 2 and 3), pZS1202 which expresses the (OVA-CD4)-MisL fusion protein from the nirB promoter (lanes 4 and 5), or pZS1204 expresses the (OVA-CD4)-MisL fusion protein from the nirB and spiC promoters in tandem (lanes 6 and 7); (B) Bovine serum albumin (lane 1), ovalbumin (lane 2), untreated (lane 3) or proteinase K-treated (lane 4) outer membrane proteins of Salmonella strain SL7207 pZ1204.

    Techniques Used: Expressing, Western Blot

    Surface exposure of (OVA-CD4)-MisL fusion protein detected by western blot analysis. Salmonella  SL7207 pZS1205 treated with 0 (lane 1), 11 (lane 2) and 33 (lane 3) μg/ml proteinase K and probed with  (A)  anti-β-lactamase pAb or  (B)  anti-OVA-CD4 peptide pAb antibodies, respectively.  (C) Salmonella  SL7207 expressing MisL fusion proteins with one (pZS1205, lanes 1 and 6), two (pZS1205-2, lanes 4 and 7) or four (pZS1205-4, lanes 5 and 8) copies of the OVA-CD4 epitope; untreated (lanes 3 to 5) or treated (lanes 6 to 8) with proteinase K. Bovine albumin (lane 1) and ovalbumin (lane 2), as negative and positive antibody controls.  (D) Salmonella  SL7207  pgtE  expressing MisL fusion proteins with one (pZS1205, lane 1), two (pZS1205-2, lane 2) or four (pZS1205-4, lane 3) copies of the OVA-CD4 epitope.
    Figure Legend Snippet: Surface exposure of (OVA-CD4)-MisL fusion protein detected by western blot analysis. Salmonella SL7207 pZS1205 treated with 0 (lane 1), 11 (lane 2) and 33 (lane 3) μg/ml proteinase K and probed with (A) anti-β-lactamase pAb or (B) anti-OVA-CD4 peptide pAb antibodies, respectively. (C) Salmonella SL7207 expressing MisL fusion proteins with one (pZS1205, lanes 1 and 6), two (pZS1205-2, lanes 4 and 7) or four (pZS1205-4, lanes 5 and 8) copies of the OVA-CD4 epitope; untreated (lanes 3 to 5) or treated (lanes 6 to 8) with proteinase K. Bovine albumin (lane 1) and ovalbumin (lane 2), as negative and positive antibody controls. (D) Salmonella SL7207 pgtE expressing MisL fusion proteins with one (pZS1205, lane 1), two (pZS1205-2, lane 2) or four (pZS1205-4, lane 3) copies of the OVA-CD4 epitope.

    Techniques Used: Western Blot, Expressing

    12) Product Images from "A bispecific immunotweezer prevents soluble PrP oligomers and abolishes prion toxicity"

    Article Title: A bispecific immunotweezer prevents soluble PrP oligomers and abolishes prion toxicity

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007335

    scPOM-bi prevents the formation of soluble, PK resistant oligomers. (A) DLS showed the presence of soluble oligomers (red shades in histograms, reported as percentage) upon addition of the POM1 toxic antibody to recombinant mPrP in vitro. Subsequent addition of POM2 did not remove the oligomers or inhibit toxicity. Smaller species comparable to monomeric forms (blue) were detected in solution when POM1 was in complex with ΔmPrP C 90-230 , lacking the flexible tail, and when POM2 was added to mPrP prior to POM1 addition. Similarly small species were found when the neuroprotective scPOM-bi was added to mPrP; the bispeficic was also capable of removing the soluble oligomers generated by POM1. DLS data is shown for 3 time points after complex formation. (n = 5 for scPOM1:mPrP, scPOM2:mPrP and scPOM-bi:mPrP; n = 3 for scPOM1:mPrP then scPOM2, scPOM2:mPrP then scPOM1 and scPOM1:mPrP then scPOM-bi) (B) DLS can only detect soluble material. To investigate the presence of insoluble aggregates we formed the mPrP:Ab complexes  in vitro , centrifuged them and analyzed the resulting supernatant with PAGE/Western blot. Soluble material was only detected in toxic combinations (POM1:mPrP or POM1:mPrP followed by POM2, red and orange). The percentage of mPrP and antibody in solution (normalized against isolated PrP or antibody) is shown; data from quantification of band intensity on SDS-PAGE (images in   S5 Fig —n = 7 for all samples tested). (C) In order to characterize both soluble oligomers and insoluble aggregates we formed the mPrP:Ab complexes  in vitro  and deposited the resulting material on microscopy slides. Confocal microscopy indicates that toxic antibody combinations (e.g. POM1:mPrP or POM1:mPrP followed by POM2, red and orange) generate species with smaller average size than protective antibody combinations. The surface area of the detected species is reported on the y axis, the horizontal line represents the average. Differences can also be appreciated by visual inspection of the confocal microscopy images (  S4 Fig —scPOM1:mPrP n = 166, scPOM2:mPrP n = 1136, scPOM-bi:mPrP n = 204, scPOM1:mPrP then scPOM2 n = 444, scPOM2:mPrP then scPOM1 n = 74 and scPOM1:mPrP then scPOM-bi n = 1767). D) The soluble oligomers generated by POM1 showed increased resistance to  in vitro  degradation by proteinase K at 2μg/ml (red). Such resistance was abolished when POM1 bound a mPrP construct lacking the FT (light red) or in non-toxic antibodies (shades of blue). Data from quantification of PK resistant bands on western blot, normalized against isolated PrP (images in   S6 Fig —scPOM1:mPrP n = 5, scPOM1:ΔPrP n = 3, scPOM2:mPrP n = 4, scPOM-bi:mPrP n = 4).
    Figure Legend Snippet: scPOM-bi prevents the formation of soluble, PK resistant oligomers. (A) DLS showed the presence of soluble oligomers (red shades in histograms, reported as percentage) upon addition of the POM1 toxic antibody to recombinant mPrP in vitro. Subsequent addition of POM2 did not remove the oligomers or inhibit toxicity. Smaller species comparable to monomeric forms (blue) were detected in solution when POM1 was in complex with ΔmPrP C 90-230 , lacking the flexible tail, and when POM2 was added to mPrP prior to POM1 addition. Similarly small species were found when the neuroprotective scPOM-bi was added to mPrP; the bispeficic was also capable of removing the soluble oligomers generated by POM1. DLS data is shown for 3 time points after complex formation. (n = 5 for scPOM1:mPrP, scPOM2:mPrP and scPOM-bi:mPrP; n = 3 for scPOM1:mPrP then scPOM2, scPOM2:mPrP then scPOM1 and scPOM1:mPrP then scPOM-bi) (B) DLS can only detect soluble material. To investigate the presence of insoluble aggregates we formed the mPrP:Ab complexes in vitro , centrifuged them and analyzed the resulting supernatant with PAGE/Western blot. Soluble material was only detected in toxic combinations (POM1:mPrP or POM1:mPrP followed by POM2, red and orange). The percentage of mPrP and antibody in solution (normalized against isolated PrP or antibody) is shown; data from quantification of band intensity on SDS-PAGE (images in S5 Fig —n = 7 for all samples tested). (C) In order to characterize both soluble oligomers and insoluble aggregates we formed the mPrP:Ab complexes in vitro and deposited the resulting material on microscopy slides. Confocal microscopy indicates that toxic antibody combinations (e.g. POM1:mPrP or POM1:mPrP followed by POM2, red and orange) generate species with smaller average size than protective antibody combinations. The surface area of the detected species is reported on the y axis, the horizontal line represents the average. Differences can also be appreciated by visual inspection of the confocal microscopy images ( S4 Fig —scPOM1:mPrP n = 166, scPOM2:mPrP n = 1136, scPOM-bi:mPrP n = 204, scPOM1:mPrP then scPOM2 n = 444, scPOM2:mPrP then scPOM1 n = 74 and scPOM1:mPrP then scPOM-bi n = 1767). D) The soluble oligomers generated by POM1 showed increased resistance to in vitro degradation by proteinase K at 2μg/ml (red). Such resistance was abolished when POM1 bound a mPrP construct lacking the FT (light red) or in non-toxic antibodies (shades of blue). Data from quantification of PK resistant bands on western blot, normalized against isolated PrP (images in S6 Fig —scPOM1:mPrP n = 5, scPOM1:ΔPrP n = 3, scPOM2:mPrP n = 4, scPOM-bi:mPrP n = 4).

    Techniques Used: Recombinant, In Vitro, Generated, Polyacrylamide Gel Electrophoresis, Western Blot, Isolation, SDS Page, Microscopy, Confocal Microscopy, Construct

    13) Product Images from "The Primary Folding Defect and Rescue of ?F508 CFTR Emerge during Translation of the Mutant Domain"

    Article Title: The Primary Folding Defect and Rescue of ?F508 CFTR Emerge during Translation of the Mutant Domain

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0015458

    Rescue of NBD1 conformation by the I539T suppressor mutation. (A) Wild-type and ΔF508 NBD1 (top panel) mRNAs containing the G550E (middle panel) or I539T (bottom panel) mutation were  in vitro  translated in the presence of  35 S-labeled methionine and cysteine and analyzed by 15% SDS-PAGE after proteinase K treatment. Asterisk indicates the 27 kDa fragment, arrowhead indicates the 25 kDa fragment. (B) Longer exposure of the 100 µg/ml proteinase K digest of  in vitro  translated NBD1, from same experiment as shown in B, showing the rescue of the 17 kDa band by the I539T but not by the G550E mutation. Gel lanes are aligned on the 25 kDa bands. (C) CFTR molecules containing the indicated mutations were  in vitro  translated, analyzed using 12% SDS-PAGE and lanes were quantified as described in   Figure 1B . The arrowhead indicates the 25 kDa fragment, which has slightly decreased mobility when the I539T mutation is present.
    Figure Legend Snippet: Rescue of NBD1 conformation by the I539T suppressor mutation. (A) Wild-type and ΔF508 NBD1 (top panel) mRNAs containing the G550E (middle panel) or I539T (bottom panel) mutation were in vitro translated in the presence of 35 S-labeled methionine and cysteine and analyzed by 15% SDS-PAGE after proteinase K treatment. Asterisk indicates the 27 kDa fragment, arrowhead indicates the 25 kDa fragment. (B) Longer exposure of the 100 µg/ml proteinase K digest of in vitro translated NBD1, from same experiment as shown in B, showing the rescue of the 17 kDa band by the I539T but not by the G550E mutation. Gel lanes are aligned on the 25 kDa bands. (C) CFTR molecules containing the indicated mutations were in vitro translated, analyzed using 12% SDS-PAGE and lanes were quantified as described in Figure 1B . The arrowhead indicates the 25 kDa fragment, which has slightly decreased mobility when the I539T mutation is present.

    Techniques Used: Mutagenesis, In Vitro, Labeling, SDS Page

    Minimal and local misfolding of ΔF508 CFTR. (A) Both CFTR and ΔF508 CFTR were translated  in vitro  in the presence of  35 S-methionine and cysteine and semi-permeabilized HT1080 cells for 60 min. Cells containing radiolabeled CFTR proteins were washed, lysed in Triton X-100, and prepared for limited proteolysis using increasing concentrations of proteinase K. The proteolytic digests were analyzed by 12% SDS-PAGE. The conformational difference between wild-type CFTR and ΔF508 CFTR is indicated by an arrowhead. (B) Relative intensities of all protease resistant fragments from a total 5 µg/ml Proteinase K digest, as in Figure 1A, were determined by total lane quantitation (Quantity One software Biorad). The y-axis represents electrophoretic mobility in 12% SDS-PA gel and the x-axis the relative intensity of the protease resistant fragments. The horizontal lines indicate the structural differences as described in A. The horizontal line indicated with an asterisk represents yet unidentified changes in the proteolytic pattern as a result of the ΔF508 mutation. The bracket represents small proteolytic fractions detected in both mutants. (C) Wild-type and ΔF508 CFTR were synthesized as in a, were subjected to 5 µg/ml proteinase K and NBD1-originated fragments were immunoprecipitated with polyclonal antibodies directed against NBD1 (Mr Pink) or against the R-region (G449). Arrowhead marks the NBD1-related fragment.
    Figure Legend Snippet: Minimal and local misfolding of ΔF508 CFTR. (A) Both CFTR and ΔF508 CFTR were translated in vitro in the presence of 35 S-methionine and cysteine and semi-permeabilized HT1080 cells for 60 min. Cells containing radiolabeled CFTR proteins were washed, lysed in Triton X-100, and prepared for limited proteolysis using increasing concentrations of proteinase K. The proteolytic digests were analyzed by 12% SDS-PAGE. The conformational difference between wild-type CFTR and ΔF508 CFTR is indicated by an arrowhead. (B) Relative intensities of all protease resistant fragments from a total 5 µg/ml Proteinase K digest, as in Figure 1A, were determined by total lane quantitation (Quantity One software Biorad). The y-axis represents electrophoretic mobility in 12% SDS-PA gel and the x-axis the relative intensity of the protease resistant fragments. The horizontal lines indicate the structural differences as described in A. The horizontal line indicated with an asterisk represents yet unidentified changes in the proteolytic pattern as a result of the ΔF508 mutation. The bracket represents small proteolytic fractions detected in both mutants. (C) Wild-type and ΔF508 CFTR were synthesized as in a, were subjected to 5 µg/ml proteinase K and NBD1-originated fragments were immunoprecipitated with polyclonal antibodies directed against NBD1 (Mr Pink) or against the R-region (G449). Arrowhead marks the NBD1-related fragment.

    Techniques Used: In Vitro, SDS Page, Quantitation Assay, Software, Mutagenesis, Synthesized, Immunoprecipitation

    The effect of ΔF508 mutation on NBD1 alone. (A) Wild-type and ΔF508 NBD1 were  in vitro  translated for 30 min, treated with indicated proteinase K concentrations as in   Figure 1 , and analyzed using 15% SDS-PAGE. The full length NBD1 domain is indicated by “#”, the asterisk (*) indicates the 27 kDa fragment, arrowhead (◂) indicates the 25 kDa fragment. (B) Similar experimental conditions as described in A, but using TPCK-trypsin as protease. A bracket (]) marks the triplet of protease resistant fragments and the dot (•) marks the 17 kDa fragment. (C) Wild-type and ΔF508 NBD1 were synthesized as in A, treated with 100 µg/ml trypsin, and fragments were immunoprecipitated with antibody 7D12 against NBD1. Fragments are labeled similar as in B. (D) Wild-type NBD1 was synthesized as in A, treated with 25 µg/ml proteinase K, and fragments were immunoprecipitated with the 7D12, 3G11 and Mr Pink antibody, recognizing specific epitopes within NBD1. Fragments are labeled similar as in A. (E) CHO cells expressing wild-type or ΔF508 NBD1 were pulse-labeled with  35 S-methionine and cysteine for 5 min and chased for indicated times. NBD1 was immunoprecipitated using polyclonal antibody Mr Pink and analyzed using 15% SDS-PAGE. NBD1 indicated by “#”. (F) Purified human wild-type and ΔF508 NBD1, indicated by “#”, were incubated with 2 µg/ml Proteinase K for 0, 2, 5, 10 and 30 minutes at room temperature. Proteolytic digests were separated using 15% SDS-PAGE and visualized by silver staining. Asterisk (*) and arrowhead (◂) indicate 27 and 25 kDa fragments resp., and are similar as in A. The open arrowhead (
    Figure Legend Snippet: The effect of ΔF508 mutation on NBD1 alone. (A) Wild-type and ΔF508 NBD1 were in vitro translated for 30 min, treated with indicated proteinase K concentrations as in Figure 1 , and analyzed using 15% SDS-PAGE. The full length NBD1 domain is indicated by “#”, the asterisk (*) indicates the 27 kDa fragment, arrowhead (◂) indicates the 25 kDa fragment. (B) Similar experimental conditions as described in A, but using TPCK-trypsin as protease. A bracket (]) marks the triplet of protease resistant fragments and the dot (•) marks the 17 kDa fragment. (C) Wild-type and ΔF508 NBD1 were synthesized as in A, treated with 100 µg/ml trypsin, and fragments were immunoprecipitated with antibody 7D12 against NBD1. Fragments are labeled similar as in B. (D) Wild-type NBD1 was synthesized as in A, treated with 25 µg/ml proteinase K, and fragments were immunoprecipitated with the 7D12, 3G11 and Mr Pink antibody, recognizing specific epitopes within NBD1. Fragments are labeled similar as in A. (E) CHO cells expressing wild-type or ΔF508 NBD1 were pulse-labeled with 35 S-methionine and cysteine for 5 min and chased for indicated times. NBD1 was immunoprecipitated using polyclonal antibody Mr Pink and analyzed using 15% SDS-PAGE. NBD1 indicated by “#”. (F) Purified human wild-type and ΔF508 NBD1, indicated by “#”, were incubated with 2 µg/ml Proteinase K for 0, 2, 5, 10 and 30 minutes at room temperature. Proteolytic digests were separated using 15% SDS-PAGE and visualized by silver staining. Asterisk (*) and arrowhead (◂) indicate 27 and 25 kDa fragments resp., and are similar as in A. The open arrowhead (

    Techniques Used: Mutagenesis, In Vitro, SDS Page, Synthesized, Immunoprecipitation, Labeling, Expressing, Purification, Incubation, Silver Staining

    14) Product Images from "Important but Differential Roles for Actin in Trafficking of Epstein-Barr Virus in B Cells and Epithelial Cells"

    Article Title: Important but Differential Roles for Actin in Trafficking of Epstein-Barr Virus in B Cells and Epithelial Cells

    Journal:

    doi: 10.1128/JVI.05883-11

    Virus DNA is lost following internalization into an epithelial cell but not a B cell. Virus was bound on ice for 2 h to Akata and Raji B cells, SVKCR2 and AGS epithelial cells, primary tonsil epithelial cells, or SVKCR2 CIITA cells which express HLA class II. Unbound virus was removed, and cells were warmed for the times indicated. Surface-bound virus was removed by digestion with proteinase K, and virus (BamK) and cellular (CRP) copies were measured by QPCR. Error bars are the standard deviations of triplicates.
    Figure Legend Snippet: Virus DNA is lost following internalization into an epithelial cell but not a B cell. Virus was bound on ice for 2 h to Akata and Raji B cells, SVKCR2 and AGS epithelial cells, primary tonsil epithelial cells, or SVKCR2 CIITA cells which express HLA class II. Unbound virus was removed, and cells were warmed for the times indicated. Surface-bound virus was removed by digestion with proteinase K, and virus (BamK) and cellular (CRP) copies were measured by QPCR. Error bars are the standard deviations of triplicates.

    Techniques Used: Real-time Polymerase Chain Reaction

    Virus DNA in the nucleus of an SVKCR2 epithelial cell remains stable. (A) Western blot of isolated nuclear and cytoplasmic fractions stained for α-tubulin or lamin B. (B) Virus was bound on ice for 2 h, unbound virus was removed, and cells were warmed for the times indicated. Surface-bound virus was removed by digestion with proteinase K. The total amount of virus DNA remaining per cell was compared with the amount in the fractionated nucleus by QPCR. Error bars are the standard deviations of triplicates.
    Figure Legend Snippet: Virus DNA in the nucleus of an SVKCR2 epithelial cell remains stable. (A) Western blot of isolated nuclear and cytoplasmic fractions stained for α-tubulin or lamin B. (B) Virus was bound on ice for 2 h, unbound virus was removed, and cells were warmed for the times indicated. Surface-bound virus was removed by digestion with proteinase K. The total amount of virus DNA remaining per cell was compared with the amount in the fractionated nucleus by QPCR. Error bars are the standard deviations of triplicates.

    Techniques Used: Western Blot, Isolation, Staining, Real-time Polymerase Chain Reaction

    15) Product Images from "Phenotypic Similarity of Transmissible Mink Encephalopathy in Cattle and L-type Bovine Spongiform Encephalopathy in a Mouse Model"

    Article Title: Phenotypic Similarity of Transmissible Mink Encephalopathy in Cattle and L-type Bovine Spongiform Encephalopathy in a Mouse Model

    Journal: Emerging Infectious Diseases

    doi: 10.3201/eid13112.070635

    Western blot analyses of protease-resistant prion protein from proteinase K–treated brain homogenates from cattle transmissible spongiform encephalopathies (TSEs). Typical bovine spongiform encephalopathy (BSE) (lanes 1, 5), L-type BSE (lane 2), transmissible mink encephalopathy (TME) in cattle (lane 3), H-type BSE (lane 4). Bars to the left of the panel indicate the 29.0- and 20.1-kDa marker positions.
    Figure Legend Snippet: Western blot analyses of protease-resistant prion protein from proteinase K–treated brain homogenates from cattle transmissible spongiform encephalopathies (TSEs). Typical bovine spongiform encephalopathy (BSE) (lanes 1, 5), L-type BSE (lane 2), transmissible mink encephalopathy (TME) in cattle (lane 3), H-type BSE (lane 4). Bars to the left of the panel indicate the 29.0- and 20.1-kDa marker positions.

    Techniques Used: Western Blot, Marker

    Western blot of protease-resistant prion protein from TgOvPrP4 mice after proteinase K digestion and immunodetection with anti-PrP Sha31 antibody. A) First passage of typical bovine spongiform encephalopathy (BSE) (lanes 2, 4, and 6) and L-type BSE (lanes 3, 5, and 7). B) First passage of TME in cattle (lanes 2, 4, and 6) and L-type (lanes 3, 5, and 7). C) Second passage of TME in cattle (lanes 2, 4, and 6) and L-type BSE (lanes 3, 5, and 7). Each lane shows PrP res  from a distinct individual mouse from each experimental group. Bars to the left of the panel indicate the 29.0- and 20.1-kDa marker positions. Lane 1, PrP res  control from a scrapie-infected TgOvPrP4 mouse (C506M3 strain).
    Figure Legend Snippet: Western blot of protease-resistant prion protein from TgOvPrP4 mice after proteinase K digestion and immunodetection with anti-PrP Sha31 antibody. A) First passage of typical bovine spongiform encephalopathy (BSE) (lanes 2, 4, and 6) and L-type BSE (lanes 3, 5, and 7). B) First passage of TME in cattle (lanes 2, 4, and 6) and L-type (lanes 3, 5, and 7). C) Second passage of TME in cattle (lanes 2, 4, and 6) and L-type BSE (lanes 3, 5, and 7). Each lane shows PrP res from a distinct individual mouse from each experimental group. Bars to the left of the panel indicate the 29.0- and 20.1-kDa marker positions. Lane 1, PrP res control from a scrapie-infected TgOvPrP4 mouse (C506M3 strain).

    Techniques Used: Western Blot, Mouse Assay, Immunodetection, Marker, Infection

    16) Product Images from "Erythrocyte G Protein as a Novel Target for Malarial Chemotherapy"

    Article Title: Erythrocyte G Protein as a Novel Target for Malarial Chemotherapy

    Journal: PLoS Medicine

    doi: 10.1371/journal.pmed.0030528

    Hematological and Signaling Characteristics and Cargo Loading of Erythrocyte Ghosts (A) Ghosts (left) were biconcave but retained less pigment than intact erythrocytes (right); bar represents 10 μm. (B) Ghost MCH (normal range for erythrocytes 27.5–33.5 pg/cell), MCV (normal range for erythrocytes 80–100 fl), and RDW (normal range for erythrocytes 11.0%–15.0%) were determined using a Coulter counter (see   Methods ). Intracellular ATP levels were determined by luciferase-based ATP assays (see   Methods ); the 95% confidence interval (CI) for ATP levels is indicated; two experiments. (C) Ghosts loaded with high molecular weight rhodamine-labeled antibody were imaged without fixation by fluorescence microscopy; bar represents 25 μm. (D) Flow cytometry of unloaded (white fill) and FITC-dextran–loaded ghosts (green fill) 2 h post-resealing, showed homogenous loading of cargo. For each cell type, 100,000 gated events were captured. (E) Western blot of GST-loaded ghosts after incubation in culture for 24 h and subsequent treatment in the presence or absence of proteinase K (Prot K) and/or Triton X-100 (TX100); see   Methods . GST was detected by GST-specific immunoblotting. (F) cAMP production in normal, intact erythrocytes treated with isopreterenol (iso) and/or propranolol (prop) as measured by enzyme-linked immunosorbent assay (see   Methods ) . Error bars show 95% CI of triplicate measurements in a representative experiment. The mean baseline cAMP concentration in control erythrocytes was 0.61 pmol cAMP (95% CI 0.20–1.01 pmol; three experiments) per 1 × 10 8  cells. Induced cAMP levels can vary—the maximal value obtained (once) was 14.58 pmol cAMP (95% CI 13.11–16.03 pmol) per 1 × 10 8  cells. (G) cAMP production in ghosts measured and depicted as described in (F). Error bars show 95% CI of triplicate measurements in a representative experiment. The mean baseline cAMP concentration in control ghosts was 1.12 pmol cAMP (95% CI 0.59–1.64 pmol; three experiments) per 1 × 10 8  cells. The maximal isoproterenol-induced value obtained in a single ghost cAMP assay was 9.00 pmol cAMP (95% CI 7.37–10.62 pmol) per 1 × 10 8  cells.
    Figure Legend Snippet: Hematological and Signaling Characteristics and Cargo Loading of Erythrocyte Ghosts (A) Ghosts (left) were biconcave but retained less pigment than intact erythrocytes (right); bar represents 10 μm. (B) Ghost MCH (normal range for erythrocytes 27.5–33.5 pg/cell), MCV (normal range for erythrocytes 80–100 fl), and RDW (normal range for erythrocytes 11.0%–15.0%) were determined using a Coulter counter (see Methods ). Intracellular ATP levels were determined by luciferase-based ATP assays (see Methods ); the 95% confidence interval (CI) for ATP levels is indicated; two experiments. (C) Ghosts loaded with high molecular weight rhodamine-labeled antibody were imaged without fixation by fluorescence microscopy; bar represents 25 μm. (D) Flow cytometry of unloaded (white fill) and FITC-dextran–loaded ghosts (green fill) 2 h post-resealing, showed homogenous loading of cargo. For each cell type, 100,000 gated events were captured. (E) Western blot of GST-loaded ghosts after incubation in culture for 24 h and subsequent treatment in the presence or absence of proteinase K (Prot K) and/or Triton X-100 (TX100); see Methods . GST was detected by GST-specific immunoblotting. (F) cAMP production in normal, intact erythrocytes treated with isopreterenol (iso) and/or propranolol (prop) as measured by enzyme-linked immunosorbent assay (see Methods ) . Error bars show 95% CI of triplicate measurements in a representative experiment. The mean baseline cAMP concentration in control erythrocytes was 0.61 pmol cAMP (95% CI 0.20–1.01 pmol; three experiments) per 1 × 10 8 cells. Induced cAMP levels can vary—the maximal value obtained (once) was 14.58 pmol cAMP (95% CI 13.11–16.03 pmol) per 1 × 10 8 cells. (G) cAMP production in ghosts measured and depicted as described in (F). Error bars show 95% CI of triplicate measurements in a representative experiment. The mean baseline cAMP concentration in control ghosts was 1.12 pmol cAMP (95% CI 0.59–1.64 pmol; three experiments) per 1 × 10 8 cells. The maximal isoproterenol-induced value obtained in a single ghost cAMP assay was 9.00 pmol cAMP (95% CI 7.37–10.62 pmol) per 1 × 10 8 cells.

    Techniques Used: Luciferase, Molecular Weight, Labeling, Fluorescence, Microscopy, Flow Cytometry, Cytometry, Western Blot, Incubation, Enzyme-linked Immunosorbent Assay, Concentration Assay, cAMP Assay

    17) Product Images from "Lesion of the Olfactory Epithelium Accelerates Prion Neuroinvasion and Disease Onset when Prion Replication Is Restricted to Neurons"

    Article Title: Lesion of the Olfactory Epithelium Accelerates Prion Neuroinvasion and Disease Onset when Prion Replication Is Restricted to Neurons

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0119863

    Western blot of prion protein in brain of hamsters following intranasal inoculation of DY TME agent in the absence and presence of a preexisting lesion to the olfactory epithelium. Brain from hamsters at sacrifice (see   Table 1 ) from vehicle (A) and methimazole (B) treatment groups followed by intranasal inoculation of DY TME were enriched for PrP Sc  by detergent extraction, differential ultracentrifugation, and proteinase K digestion. For each sample 20 mg tissue equivalents was examined by Western blot for PrP Sc , except for two samples in which only 2 mg tissue equivalents was used (B, lanes 1 and 5). None of the hamsters in the vehicle group and only one hamster in the methimazole group exhibited symptoms of DY TME (B, lane 5 and   Table 1 ). The hamster exhibiting clinical symptoms had a strong PrP Sc  signal in brain, and two additional hamsters in the methimazole group that were clinically normal and sacrificed after 414 days postinoculation also had evidence of PrP Sc  in brain (B, lanes 1 and 7). Molecular weight markers at edge of western blots correspond to 20, 30, and 40 kilodaltons.
    Figure Legend Snippet: Western blot of prion protein in brain of hamsters following intranasal inoculation of DY TME agent in the absence and presence of a preexisting lesion to the olfactory epithelium. Brain from hamsters at sacrifice (see Table 1 ) from vehicle (A) and methimazole (B) treatment groups followed by intranasal inoculation of DY TME were enriched for PrP Sc by detergent extraction, differential ultracentrifugation, and proteinase K digestion. For each sample 20 mg tissue equivalents was examined by Western blot for PrP Sc , except for two samples in which only 2 mg tissue equivalents was used (B, lanes 1 and 5). None of the hamsters in the vehicle group and only one hamster in the methimazole group exhibited symptoms of DY TME (B, lane 5 and Table 1 ). The hamster exhibiting clinical symptoms had a strong PrP Sc signal in brain, and two additional hamsters in the methimazole group that were clinically normal and sacrificed after 414 days postinoculation also had evidence of PrP Sc in brain (B, lanes 1 and 7). Molecular weight markers at edge of western blots correspond to 20, 30, and 40 kilodaltons.

    Techniques Used: Western Blot, Molecular Weight

    Western blot of prion protein in brain, olfactory bulb, and nasal turbinate of mice following intranasal inoculation of prions in the absence and presence of a preexisting lesion to the olfactory epithelium. Immunodetection of the prion protein in tissues from C57Bl/6 mice (A, B) and HPrP7752KO transgenic mice (C, D) following intranasal inoculation of RML scrapie and HY TME, respectively. Mice were pretreated with vehicle (veh) and methimazole (mmi) 48 hours prior to prion inoculation. Clinically ill C57Bl/6 mice all had PrP Sc  deposition in brain (not shown), olfactory bulb (A), and nasal turbinate (B) following proteinase K (PK) digestion of tissue homogenates (A) and PrP Sc  enrichment methods that included a PK digestion step (B). In clinically ill HPrP7752KO mice, PrP Sc  was detected in brain (C), olfactory bulb (not shown), and in  > 75% of nasal turbinates (D) following PrP Sc  enrichment. Asterisk (C, D) indicates a mouse that did not develop clinical symptoms of prion disease and was devoid of PrP Sc . In non-proteinase K (non-PK) treated samples, 20 μg protein from brain (C) and 40 μg protein from nasal turbinate (D) were analyzed while for PK treated samples, 100 μg protein from brain (C) and 1 mg protein from nasal turbinate (D) were used as starting material for PrP Sc  enrichment and analysis. RML scrapie-infected brain (Br) control is indicated.
    Figure Legend Snippet: Western blot of prion protein in brain, olfactory bulb, and nasal turbinate of mice following intranasal inoculation of prions in the absence and presence of a preexisting lesion to the olfactory epithelium. Immunodetection of the prion protein in tissues from C57Bl/6 mice (A, B) and HPrP7752KO transgenic mice (C, D) following intranasal inoculation of RML scrapie and HY TME, respectively. Mice were pretreated with vehicle (veh) and methimazole (mmi) 48 hours prior to prion inoculation. Clinically ill C57Bl/6 mice all had PrP Sc deposition in brain (not shown), olfactory bulb (A), and nasal turbinate (B) following proteinase K (PK) digestion of tissue homogenates (A) and PrP Sc enrichment methods that included a PK digestion step (B). In clinically ill HPrP7752KO mice, PrP Sc was detected in brain (C), olfactory bulb (not shown), and in > 75% of nasal turbinates (D) following PrP Sc enrichment. Asterisk (C, D) indicates a mouse that did not develop clinical symptoms of prion disease and was devoid of PrP Sc . In non-proteinase K (non-PK) treated samples, 20 μg protein from brain (C) and 40 μg protein from nasal turbinate (D) were analyzed while for PK treated samples, 100 μg protein from brain (C) and 1 mg protein from nasal turbinate (D) were used as starting material for PrP Sc enrichment and analysis. RML scrapie-infected brain (Br) control is indicated.

    Techniques Used: Western Blot, Mouse Assay, Immunodetection, Transgenic Assay, Infection

    18) Product Images from "A30P ?-Synuclein interferes with the stable integration of adult-born neurons into the olfactory network"

    Article Title: A30P ?-Synuclein interferes with the stable integration of adult-born neurons into the olfactory network

    Journal: Scientific Reports

    doi: 10.1038/srep03931

    Olfactory bulb pathology in A30P α-SYN mice. (a) Immunofluorescent micrographs showing the overexpression pattern of human A30P α-SYN (15G7, red) under control of the Thy1-promoter in the OB of 6-month-old transgenic mice: z-stack projections of confocal series. Scale bar - 50 μm. (b–e) Representative OB sections immunostained for phosphorylated α-SYN (pSer129, DAB), a marker for aberrant protein modification. Scale bars - 50 μm. (b) Distribution of phosphorylated α-SYN in (c) PGNs, (d) MCs and (e) GCs. Note that only MCs are positive for pSer129. (f–j) Representative OB sections with immunostaining for fibrillar protein, detected by Proteinase K digestion and human α-SYN specific antibody (15G7, DAB). Scale bars - 50 μm. Images with higher magnification of the (g) glomerular, (h) MC and (i) GC layer. Some α-SYN aggregates were detected in MCs of transgenic, but not wild-type, mice. (j) Olfactory discrimination task. Six-month-old Control (wild-type) ( n  = 8) and A30P α-SYN mice ( n  = 7) were exposed to different binary odour mixtures (e.g. 55% S +  and 45% S − vs . 45% S +  and 55% S − ). Mice were trained to dig in a bowl containing more of S +  odour. Correct choices for S +  were plotted against the different mixture pairs. Note that with increasing similarity of the mixtures, the performance of A30P α-SYN mice dropped compared to Control. (k) Olfactory memory task. ** p
    Figure Legend Snippet: Olfactory bulb pathology in A30P α-SYN mice. (a) Immunofluorescent micrographs showing the overexpression pattern of human A30P α-SYN (15G7, red) under control of the Thy1-promoter in the OB of 6-month-old transgenic mice: z-stack projections of confocal series. Scale bar - 50 μm. (b–e) Representative OB sections immunostained for phosphorylated α-SYN (pSer129, DAB), a marker for aberrant protein modification. Scale bars - 50 μm. (b) Distribution of phosphorylated α-SYN in (c) PGNs, (d) MCs and (e) GCs. Note that only MCs are positive for pSer129. (f–j) Representative OB sections with immunostaining for fibrillar protein, detected by Proteinase K digestion and human α-SYN specific antibody (15G7, DAB). Scale bars - 50 μm. Images with higher magnification of the (g) glomerular, (h) MC and (i) GC layer. Some α-SYN aggregates were detected in MCs of transgenic, but not wild-type, mice. (j) Olfactory discrimination task. Six-month-old Control (wild-type) ( n = 8) and A30P α-SYN mice ( n = 7) were exposed to different binary odour mixtures (e.g. 55% S + and 45% S − vs . 45% S + and 55% S − ). Mice were trained to dig in a bowl containing more of S + odour. Correct choices for S + were plotted against the different mixture pairs. Note that with increasing similarity of the mixtures, the performance of A30P α-SYN mice dropped compared to Control. (k) Olfactory memory task. ** p

    Techniques Used: Mouse Assay, Over Expression, Transgenic Assay, Marker, Modification, Immunostaining, Gas Chromatography

    19) Product Images from "Structural and functional properties of prefibrillar α-synuclein oligomers"

    Article Title: Structural and functional properties of prefibrillar α-synuclein oligomers

    Journal: Scientific Reports

    doi: 10.1038/srep24526

    Seeded aggregation of reporter ChFP-α-syn by exogenous α-syn assemblies assessed by increased resistance to proteolysis. Western blot analysis of the ChFP-α-syn resistance to proteinase K in lysates from Neuro2A cells exposed for 24 h to 0.3 nM α-syn fibrils, equivalent to 2.5 μM monomeric α-syn ( a ), 300 nM large GA-cross-linked α-syn oligomers, equivalent to 5 μM monomeric α-syn ( b ), or 5 μM monomeric α-syn ( c ). The lysates (40 μl corresponding to ~80000 cells), were incubated in the presence of the indicated concentrations of proteinase K for 20 min at 37 °C. The proteolytic reactions were stopped by addition of 1 mM PMSF and immediate denaturation in Laemmli buffer for 5 min at 95 °C. The samples were analyzed on 12% Tris-Glycine SDS-PAGE. ChFP–α-syn ( a – c ) was probed with mouse monoclonal anti-α-syn antibody (BD Biosciences Cat #610787). The immunoreactivity of α-tubulin (mouse monoclonal antibody DM1A, Abcam Cat #ab7291) in the initial lysate was used as a loading control ( d ). ChFP-α-syn assemblies seeded by α-syn fibrils resisted 0.1 μg/ml proteinase K ( a ). ChFP-α-syn from cells exposed to monomeric α-syn was fully degraded by 0.01 μg/ml proteinase K ( c ). ChFP-α-syn originating from cells exposed to large GA-cross-linked oligomers resisted 0.01 μg/ml and was fully degraded by 0.05 μg/ml proteinase K ( b ).
    Figure Legend Snippet: Seeded aggregation of reporter ChFP-α-syn by exogenous α-syn assemblies assessed by increased resistance to proteolysis. Western blot analysis of the ChFP-α-syn resistance to proteinase K in lysates from Neuro2A cells exposed for 24 h to 0.3 nM α-syn fibrils, equivalent to 2.5 μM monomeric α-syn ( a ), 300 nM large GA-cross-linked α-syn oligomers, equivalent to 5 μM monomeric α-syn ( b ), or 5 μM monomeric α-syn ( c ). The lysates (40 μl corresponding to ~80000 cells), were incubated in the presence of the indicated concentrations of proteinase K for 20 min at 37 °C. The proteolytic reactions were stopped by addition of 1 mM PMSF and immediate denaturation in Laemmli buffer for 5 min at 95 °C. The samples were analyzed on 12% Tris-Glycine SDS-PAGE. ChFP–α-syn ( a – c ) was probed with mouse monoclonal anti-α-syn antibody (BD Biosciences Cat #610787). The immunoreactivity of α-tubulin (mouse monoclonal antibody DM1A, Abcam Cat #ab7291) in the initial lysate was used as a loading control ( d ). ChFP-α-syn assemblies seeded by α-syn fibrils resisted 0.1 μg/ml proteinase K ( a ). ChFP-α-syn from cells exposed to monomeric α-syn was fully degraded by 0.01 μg/ml proteinase K ( c ). ChFP-α-syn originating from cells exposed to large GA-cross-linked oligomers resisted 0.01 μg/ml and was fully degraded by 0.05 μg/ml proteinase K ( b ).

    Techniques Used: Western Blot, Incubation, SDS Page

    20) Product Images from "UK Iatrogenic Creutzfeldt–Jakob disease: investigating human prion transmission across genotypic barriers using human tissue-based and molecular approaches"

    Article Title: UK Iatrogenic Creutzfeldt–Jakob disease: investigating human prion transmission across genotypic barriers using human tissue-based and molecular approaches

    Journal: Acta Neuropathologica

    doi: 10.1007/s00401-016-1638-x

    PrP res  type found in the iCJD cases examined.  a  Four PrP res  molecular types were detected by Western blot analysis of proteinase K treated brain extracts of hGH-iCJD patients; type 1, type i, type i + 2 and type 2. Representative blots of each PrP res  type are shown. Each sample ( middle lane ) is flanked by type 1 (T1) ( left lane ) and type 2 (T2) ( right lane ) reference standards from sCJD MM1 and VV2 subtype cases, respectively. Case number and codon 129  PRNP  genotype are indicated above each blot, whilst PrP res  type is indicated below. The most informative exposure for the test and reference standard samples from a series of timed exposures is shown. Blots were probed using the monoclonal antibody 3F4.  b  Western blot analysis of proteinase K treated samples of PrP res  types detected in hGH-iCJD patients using the monoclonal antibody 12B2 which detects type 1 PrP res  and shown after a prolonged (30 min) and abbreviated (3 min) exposure time. Approximate molecular mass is shown in kDa. The immunoblots shown are representative of at least three technical repeats
    Figure Legend Snippet: PrP res type found in the iCJD cases examined. a Four PrP res molecular types were detected by Western blot analysis of proteinase K treated brain extracts of hGH-iCJD patients; type 1, type i, type i + 2 and type 2. Representative blots of each PrP res type are shown. Each sample ( middle lane ) is flanked by type 1 (T1) ( left lane ) and type 2 (T2) ( right lane ) reference standards from sCJD MM1 and VV2 subtype cases, respectively. Case number and codon 129 PRNP genotype are indicated above each blot, whilst PrP res type is indicated below. The most informative exposure for the test and reference standard samples from a series of timed exposures is shown. Blots were probed using the monoclonal antibody 3F4. b Western blot analysis of proteinase K treated samples of PrP res types detected in hGH-iCJD patients using the monoclonal antibody 12B2 which detects type 1 PrP res and shown after a prolonged (30 min) and abbreviated (3 min) exposure time. Approximate molecular mass is shown in kDa. The immunoblots shown are representative of at least three technical repeats

    Techniques Used: Western Blot

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    Article Snippet: Briefly, RNase A was dissolved in 10 mM Sodium acetate (pH 5.2) to a final concentration of 10 mg/ml and incubated in boiling water for 15 min to inactivate any residual DNase activity. .. Proteinase K was from Roche Applied Science.

    Article Title: Agent strain variation in human prion disease: insights from a molecular and pathological review of the National Institutes of Health series of experimentally transmitted disease
    Article Snippet: Preparation of samples including PrPTSE purification, western blotting and PrPTSE typing were performed according to established methods ( , ; , ). .. In particular, all samples were homogenized in lysis buffer plus [100 mmol/l NaCl, 10 mmol/l EDTA, 0.5% (v/v) Nonidet P 40, 0.5% (w/v) sodium deoxycholate, 100 mmol/l Tris–HCl, pH 6.9] and digested with proteinase K (Roche Diagnostics, specific activity by certificate of analysis: 47.9 U/mg) at a final concentration of 5 U/ml. .. Purified PrPTSE was obtained from about 400 mg of tissue from the cerebral cortex, which was subjected to three cycles of sarkosyl extraction and differential centrifugation to yield the P3 pellet.

    Western Blot:

    Article Title: Dissociation of Infectivity from Seeding Ability in Prions with Alternate Docking Mechanism
    Article Snippet: 10 µL of each reaction product was transferred to fresh brain homogenate for the following round. .. Mouse PrPSc was detected by digestion with 25 µg/mL proteinase K (Roche, Indianapolis, IN) for 30 min. at 37°C and 750 r.p.m. shaking, polyacrylamide gel electrophoresis (PAGE), transfer to polyvinylidene fluoride (PVDF), and Western detection with anti-PrP and horseradish peroxidase-conjugated anti-mouse sheep IgG (GE Healthcare, Piscataway, NJ). .. Signals were detected by enhanced chemiluminescence (ECL) (SuperSignal West Femto Substrate, Pierce, Rockford, IL) and visualized by a Fuji (Fujifilm) LAS-3000 chemiluminescence documentation system.

    Article Title: Detection of protease-resistant cervid prion protein in water from a CWD-endemic area
    Article Snippet: Paragraph title: PK digestion and western blot (WB). ... Amplified samples were treated with 50 µg/ml of Proteinase-K (Roche) for 30 min at 45°C and PK was inactivated by the addition of lithium dodecyl sulfate loading buffer (Invitrogen) and heat inactivation for five minutes at 99°C.

    Article Title: Agent strain variation in human prion disease: insights from a molecular and pathological review of the National Institutes of Health series of experimentally transmitted disease
    Article Snippet: Preparation of samples including PrPTSE purification, western blotting and PrPTSE typing were performed according to established methods ( , ; , ). .. In particular, all samples were homogenized in lysis buffer plus [100 mmol/l NaCl, 10 mmol/l EDTA, 0.5% (v/v) Nonidet P 40, 0.5% (w/v) sodium deoxycholate, 100 mmol/l Tris–HCl, pH 6.9] and digested with proteinase K (Roche Diagnostics, specific activity by certificate of analysis: 47.9 U/mg) at a final concentration of 5 U/ml.

    Article Title: Evolutionary conservation and in vitro reconstitution of microsporidian iron–sulfur cluster biosynthesis
    Article Snippet: The 25,000g pellet was washed twice with HSDP buffer without proteinase inhibitors and incubated with 50 μg ml−1 proteinase K (Roche) for 20 min, or 0.2% (v/v) Triton X-100 for 10 min at room temperature followed by incubation with 50 μg ml−1 proteinase K (Roche) for 20 min. .. The membrane fractions were then incubated with trichloroacetic acid at 20% (v/v) final concentration for 30 min on ice.

    Article Title: Improved delivery of the OVA-CD4 peptide to T helper cells by polymeric surface display on Salmonella
    Article Snippet: Proteinase K (recombinant, Roche Diagnostics GmbH, Mannheim, Germany) was added to final concentrations of 11, 33 or 100 μg/ml, leaving one sample as negative control. .. The samples were incubated at 37°C for 30 min, and protease activity was stopped by adding AEBSF [4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride] to each sample at final concentration of 0.5 mM.

    Article Title: Acquired transmissibility of sheep-passaged L-type bovine spongiform encephalopathy prion to wild-type mice
    Article Snippet: Paragraph title: Western Blotting (WB) ... The sample was then digested with 40 μg/mL proteinase K (PK; Roche Diagnostics, Basel, Switzerland) and the digestion was terminated using 2 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (Pefabloc; Roche Diagnostics).

    Article Title: Role of Cyclophilin A from Brains of Prion-infected Mice in Stimulation of Cytokine Release by Microglia and Astroglia in Vitro
    Article Snippet: Paragraph title: Western Blots and Antibodies ... Samples were treated with 50 μg/ml proteinase K (Roche Applied Science) for 45 min at 37 °C.

    Article Title: Lesion of the Olfactory Epithelium Accelerates Prion Neuroinvasion and Disease Onset when Prion Replication Is Restricted to Neurons
    Article Snippet: Paragraph title: PrPSc enrichment and western blot ... For detection of PrPSc in brain and olfactory bulb homogenates, protein (100 to 300 μg) from tissues of clinically ill rodents was digested with proteinase K (Roche Diagnostics Corporation, Indianapolis, IN) at 10 μg/ml at a protein sample concentration of 1 mg/ml and incubated at 37°C for one hour with constant agitation followed by the addition of 1 mM PefaBloc (Roche Diagnostics Corporation, Indianapolis, IN).

    Derivative Assay:

    Article Title: Role of Cyclophilin A from Brains of Prion-infected Mice in Stimulation of Cytokine Release by Microglia and Astroglia in Vitro
    Article Snippet: To determine which bands were CyPA-specific, in some experiments prior to immunoblotting, α-CyPA was preincubated overnight at 4 °C with excess peptide corresponding to the epitope recognized by the antibody (synthetic peptide derived from residues 100–165) (Abcam). .. Samples were treated with 50 μg/ml proteinase K (Roche Applied Science) for 45 min at 37 °C.

    Protease Inhibitor:

    Article Title: Hepatitis C Virus p7 is Critical for Capsid Assembly and Envelopment
    Article Snippet: Subsequently, 50 µl of the crude lysate was left untreated, while 50 µl was treated with 50 µg/ml proteinase K (Roche, Mannheim, Germany) for 1 h on ice and another 50 µl was lysed with 5% (v/v) Triton X-100 prior to proteinase K treatment. .. The amount of residual core protein was determined by SDS-PAGE and immunoblotting.

    Article Title: Lesion of the Olfactory Epithelium Accelerates Prion Neuroinvasion and Disease Onset when Prion Replication Is Restricted to Neurons
    Article Snippet: Frozen brain, olfactory bulb and nasal turbinate were homogenized in lysis buffer (i.e., 10 mM Tris-HCL, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% sodium deoxycholate and 0.5% ipegal) containing 1X complete protease inhibitor (Roche Diagnostics, Indianapolis, IN) to approximately 10% (weight per volume). .. For detection of PrPSc in brain and olfactory bulb homogenates, protein (100 to 300 μg) from tissues of clinically ill rodents was digested with proteinase K (Roche Diagnostics Corporation, Indianapolis, IN) at 10 μg/ml at a protein sample concentration of 1 mg/ml and incubated at 37°C for one hour with constant agitation followed by the addition of 1 mM PefaBloc (Roche Diagnostics Corporation, Indianapolis, IN).

    Infection:

    Article Title: Rapid, high-throughput detection of PrPSc by 96-well immunoassay
    Article Snippet: Brain tissue was collected from Syrian golden hamsters infected with either the hyper (HY) or drowsy (DY) strains of transmissible mink encephalopathy (TME) agent at terminal disease or uninfected control hamsters. .. Brain tissue was homogenized to 10% w/v in Dulbecco's phosphate buffered saline (DPBS; Mediatech, Herndon, VA) and was incubated with an equal volume of either proteinase K (final concentration 4 U/ml PK; Roche Diagnostics GmbH, Mannheim, Germany) at 37°C for 1 hour with constant agitation, or diluted in DPBS for undigested controls.

    Article Title: Evolutionary conservation and in vitro reconstitution of microsporidian iron–sulfur cluster biosynthesis
    Article Snippet: Proteinase K protection assays were performed on the 25,000g (mitosomal-enriched) pellet of infected RK-13 cells obtained as described above. .. The 25,000g pellet was washed twice with HSDP buffer without proteinase inhibitors and incubated with 50 μg ml−1 proteinase K (Roche) for 20 min, or 0.2% (v/v) Triton X-100 for 10 min at room temperature followed by incubation with 50 μg ml−1 proteinase K (Roche) for 20 min.

    Imaging:

    Article Title: Chromatin architecture may dictate the target site for DMC1, but not for RAD51, during homologous pairing
    Article Snippet: The reaction mixtures were further incubated at 37 °C for 10 min, and the reactions were stopped by the addition of 2 μl of stop solution, containing SDS (0.2%) and proteinase K (1.4 mg/ml, Roche Applied Science). .. The resulting DNA products were analyzed by 1% agarose gel electrophoresis, in 1× TAE buffer at 4 V/cm for 2 h. The gels were dried and exposed to an imaging plate.

    Article Title: Chromatin architecture may dictate the target site for DMC1, but not for RAD51, during homologous pairing
    Article Snippet: After a 10 min incubation at 37 °C, 1 μl of naked dsDNA (final 30 μM in nucleotides) was added. .. The reactions were stopped by the addition of 2 μl of stop solution, containing SDS (0.2%) and proteinase K (1.4 mg/ml, Roche Applied Science), and the DNA products were analyzed by 1% agarose gel electrophoresis, in 1× TAE buffer at 4 V/cm for 2 h. The gels were dried and exposed to an imaging plate. .. The gel images were visualized using an FLA-7000 imaging analyzer (Fujifilm), and the band intensities were quantitated with the Multi Gauge software (Fujifilm).

    Protein Concentration:

    Article Title: Lesion of the Olfactory Epithelium Accelerates Prion Neuroinvasion and Disease Onset when Prion Replication Is Restricted to Neurons
    Article Snippet: The protein concentration in tissue homogenates was determined using the micro-BCA assay (Pierce Protein Research, Rockford, IL). .. For detection of PrPSc in brain and olfactory bulb homogenates, protein (100 to 300 μg) from tissues of clinically ill rodents was digested with proteinase K (Roche Diagnostics Corporation, Indianapolis, IN) at 10 μg/ml at a protein sample concentration of 1 mg/ml and incubated at 37°C for one hour with constant agitation followed by the addition of 1 mM PefaBloc (Roche Diagnostics Corporation, Indianapolis, IN).

    Polymerase Chain Reaction:

    Article Title: Prion Pathogenesis in the Absence of NLRP3/ASC Inflammasomes
    Article Snippet: Mice were kept in a conventional hygienic grade facility, housed in groups of 3–10 in conventional type II and type III cages, under a 12 h light/12 h dark cycle (from 7 am to 7 pm) at 22±1°C, with unrestricted access to sterilized food (Kliba No. 3340, Provimi Kliba, Kaiseraugst, Switzerland) and water. .. Tissue biopsies were digested in 10 mM Tris pH 9, 50 mM KCl, 0.5% Tween20, 0.5% NP40, 0.1 mg/mL proteinase K (Roche) and 1 µL of crude digest was employed as template for a PCR reaction using the Red Taq ReadyMix PCR reaction Mix (Sigma) on a GeneAmp PCR System 9700 (Applied Biosystems). .. For the Nlrp3 tm1Ts c allele, 0.33 μM of each of the following primers were used (5’→3’): AAG TCG TGC TGC TTC ATG T, TCA AGC TAA GAG AAC TTT CTG and ACA CTC GTC ATC TTC AGC A.

    Recombinant:

    Article Title: Improved delivery of the OVA-CD4 peptide to T helper cells by polymeric surface display on Salmonella
    Article Snippet: Bacteria grown to log phase (A600 ~ 0.6) were washed in PBS three times, then divided in four samples. .. Proteinase K (recombinant, Roche Diagnostics GmbH, Mannheim, Germany) was added to final concentrations of 11, 33 or 100 μg/ml, leaving one sample as negative control. .. The samples were incubated at 37°C for 30 min, and protease activity was stopped by adding AEBSF [4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride] to each sample at final concentration of 0.5 mM.

    Article Title: Role of Cyclophilin A from Brains of Prion-infected Mice in Stimulation of Cytokine Release by Microglia and Astroglia in Vitro
    Article Snippet: Purified recombinant human CyPA (rhCyPA) from two different lots (Abcam lot numbers 784230 and 838900) was used as a control in immunoblots and glial stimulation experiments as well as a source to purify truncated CyPA forms. .. Samples were treated with 50 μg/ml proteinase K (Roche Applied Science) for 45 min at 37 °C.

    Nucleic Acid Electrophoresis:

    Article Title: Structural and functional characterization of two alpha-synuclein strains
    Article Snippet: α-syn fibrils and ribbons (1.4 mg ml−1 ) in 20 mM Tris-HCl, pH 7.5, 1 mM EGTA were treated at 37 °C by Proteinase K (3.8 μg ml−1 ) (Roche). .. α-syn fibrils and ribbons (1.4 mg ml−1 ) in 20 mM Tris-HCl, pH 7.5, 1 mM EGTA were treated at 37 °C by Proteinase K (3.8 μg ml−1 ) (Roche).

    Size-exclusion Chromatography:

    Article Title: Prion Pathogenesis in the Absence of NLRP3/ASC Inflammasomes
    Article Snippet: Tissue biopsies were digested in 10 mM Tris pH 9, 50 mM KCl, 0.5% Tween20, 0.5% NP40, 0.1 mg/mL proteinase K (Roche) and 1 µL of crude digest was employed as template for a PCR reaction using the Red Taq ReadyMix PCR reaction Mix (Sigma) on a GeneAmp PCR System 9700 (Applied Biosystems). .. For the Nlrp3 tm1Ts c allele, 0.33 μM of each of the following primers were used (5’→3’): AAG TCG TGC TGC TTC ATG T, TCA AGC TAA GAG AAC TTT CTG and ACA CTC GTC ATC TTC AGC A.

    Purification:

    Article Title: Proteomic Characterization of Pseudorabies Virus Extracellular Virions
    Article Snippet: Paragraph title: Virion purification. ... For protease treatment of virions, virions were resuspended in 1 ml of MNT buffer containing 10 μg/ml proteinase K (Roche, Mannheim, Germany) for 45 min at room temperature and subsequently treated with 2 mM phenylmethylsulfonyl fluoride (PMSF; Roche) prior to density gradient centrifugation ( ).

    Article Title: Agent strain variation in human prion disease: insights from a molecular and pathological review of the National Institutes of Health series of experimentally transmitted disease
    Article Snippet: Preparation of samples including PrPTSE purification, western blotting and PrPTSE typing were performed according to established methods ( , ; , ). .. In particular, all samples were homogenized in lysis buffer plus [100 mmol/l NaCl, 10 mmol/l EDTA, 0.5% (v/v) Nonidet P 40, 0.5% (w/v) sodium deoxycholate, 100 mmol/l Tris–HCl, pH 6.9] and digested with proteinase K (Roche Diagnostics, specific activity by certificate of analysis: 47.9 U/mg) at a final concentration of 5 U/ml.

    Article Title: Role of Cyclophilin A from Brains of Prion-infected Mice in Stimulation of Cytokine Release by Microglia and Astroglia in Vitro
    Article Snippet: Purified recombinant human CyPA (rhCyPA) from two different lots (Abcam lot numbers 784230 and 838900) was used as a control in immunoblots and glial stimulation experiments as well as a source to purify truncated CyPA forms. .. Samples were treated with 50 μg/ml proteinase K (Roche Applied Science) for 45 min at 37 °C.

    Article Title: Transglutaminase-mediated Intramolecular Cross-linking of Membrane-bound ?-Synuclein Promotes Amyloid Formation in Lewy Bodies
    Article Snippet: Total brain tissue proteins were purified after TriPure lysis and merging TriPure-soluble and insoluble fractions. .. Sample proteins were supplemented with ∼ 0.1 pmol of GGEL (Sigma) as internal (light) standard, subjected to limited acid hydrolysis (0.5 n trifluoroacetic acid) at 95 °C for 16 h), dried, and then digested with proteinase K (Roche Applied Science, 20 μg/ml in 10 m m HEPES-Na, pH 8.0, and 1 m m CaCl2 ) in water containing 20% H2 18 O (Aldrich) at 56 °C overnight.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Dissociation of Infectivity from Seeding Ability in Prions with Alternate Docking Mechanism
    Article Snippet: 10 µL of each reaction product was transferred to fresh brain homogenate for the following round. .. Mouse PrPSc was detected by digestion with 25 µg/mL proteinase K (Roche, Indianapolis, IN) for 30 min. at 37°C and 750 r.p.m. shaking, polyacrylamide gel electrophoresis (PAGE), transfer to polyvinylidene fluoride (PVDF), and Western detection with anti-PrP and horseradish peroxidase-conjugated anti-mouse sheep IgG (GE Healthcare, Piscataway, NJ). .. Signals were detected by enhanced chemiluminescence (ECL) (SuperSignal West Femto Substrate, Pierce, Rockford, IL) and visualized by a Fuji (Fujifilm) LAS-3000 chemiluminescence documentation system.

    Article Title: Structural and functional characterization of two alpha-synuclein strains
    Article Snippet: α-syn fibrils and ribbons (1.4 mg ml−1 ) in 20 mM Tris-HCl, pH 7.5, 1 mM EGTA were treated at 37 °C by Proteinase K (3.8 μg ml−1 ) (Roche). .. α-syn fibrils and ribbons (1.4 mg ml−1 ) in 20 mM Tris-HCl, pH 7.5, 1 mM EGTA were treated at 37 °C by Proteinase K (3.8 μg ml−1 ) (Roche).

    Sample Prep:

    Article Title: Rapid, high-throughput detection of PrPSc by 96-well immunoassay
    Article Snippet: Paragraph title: Sample preparation. ... Brain tissue was homogenized to 10% w/v in Dulbecco's phosphate buffered saline (DPBS; Mediatech, Herndon, VA) and was incubated with an equal volume of either proteinase K (final concentration 4 U/ml PK; Roche Diagnostics GmbH, Mannheim, Germany) at 37°C for 1 hour with constant agitation, or diluted in DPBS for undigested controls.

    SDS Page:

    Article Title: Improved delivery of the OVA-CD4 peptide to T helper cells by polymeric surface display on Salmonella
    Article Snippet: Proteinase K (recombinant, Roche Diagnostics GmbH, Mannheim, Germany) was added to final concentrations of 11, 33 or 100 μg/ml, leaving one sample as negative control. .. The samples were incubated at 37°C for 30 min, and protease activity was stopped by adding AEBSF [4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride] to each sample at final concentration of 0.5 mM.

    Article Title: Hepatitis C Virus p7 is Critical for Capsid Assembly and Envelopment
    Article Snippet: Subsequently, 50 µl of the crude lysate was left untreated, while 50 µl was treated with 50 µg/ml proteinase K (Roche, Mannheim, Germany) for 1 h on ice and another 50 µl was lysed with 5% (v/v) Triton X-100 prior to proteinase K treatment. .. Proteinase K digestion was terminated by addition of PMSF (phenylmethylsulfonyl fluoride; AppliChem) at a final concentration of 5 mM and incubation of the sample on ice for 10 min.

    Software:

    Article Title: Chromatin architecture may dictate the target site for DMC1, but not for RAD51, during homologous pairing
    Article Snippet: The reaction mixtures were further incubated at 37 °C for 10 min, and the reactions were stopped by the addition of 2 μl of stop solution, containing SDS (0.2%) and proteinase K (1.4 mg/ml, Roche Applied Science). .. The resulting DNA products were analyzed by 1% agarose gel electrophoresis, in 1× TAE buffer at 4 V/cm for 2 h. The gels were dried and exposed to an imaging plate.

    Electrophoresis:

    Article Title: Acquired transmissibility of sheep-passaged L-type bovine spongiform encephalopathy prion to wild-type mice
    Article Snippet: The sample was then digested with 40 μg/mL proteinase K (PK; Roche Diagnostics, Basel, Switzerland) and the digestion was terminated using 2 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (Pefabloc; Roche Diagnostics). .. The sample was then digested with 40 μg/mL proteinase K (PK; Roche Diagnostics, Basel, Switzerland) and the digestion was terminated using 2 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (Pefabloc; Roche Diagnostics).

    Negative Control:

    Article Title: Improved delivery of the OVA-CD4 peptide to T helper cells by polymeric surface display on Salmonella
    Article Snippet: Bacteria grown to log phase (A600 ~ 0.6) were washed in PBS three times, then divided in four samples. .. Proteinase K (recombinant, Roche Diagnostics GmbH, Mannheim, Germany) was added to final concentrations of 11, 33 or 100 μg/ml, leaving one sample as negative control. .. The samples were incubated at 37°C for 30 min, and protease activity was stopped by adding AEBSF [4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride] to each sample at final concentration of 0.5 mM.

    Positron Emission Tomography:

    Article Title: A Fluorescent Oligothiophene-Bis-Triazine ligand interacts with PrP fibrils and detects SDS-resistant oligomers in human prion diseases
    Article Snippet: Pefabloc and proteinase K were purchased from Roche Diagnostics (Mannheim, Germany). .. Pefabloc and proteinase K were purchased from Roche Diagnostics (Mannheim, Germany).

    Agarose Gel Electrophoresis:

    Article Title: Chromatin architecture may dictate the target site for DMC1, but not for RAD51, during homologous pairing
    Article Snippet: After a 10 min incubation at 37 °C, 1 μl of naked dsDNA (final 30 μM in nucleotides) was added. .. The reactions were stopped by the addition of 2 μl of stop solution, containing SDS (0.2%) and proteinase K (1.4 mg/ml, Roche Applied Science), and the DNA products were analyzed by 1% agarose gel electrophoresis, in 1× TAE buffer at 4 V/cm for 2 h. The gels were dried and exposed to an imaging plate. .. The gel images were visualized using an FLA-7000 imaging analyzer (Fujifilm), and the band intensities were quantitated with the Multi Gauge software (Fujifilm).

    In Vitro:

    Article Title: The Primary Folding Defect and Rescue of ?F508 CFTR Emerge during Translation of the Mutant Domain
    Article Snippet: Folding of CFTR and NBD1 was assayed through protease susceptibility. .. Immediately after in vitro translation, proteins were subjected to increasing concentrations of TPCK-trypsin (Sigma) or Proteinase K (Roche) as described before . .. Experiments were performed using different batches of Proteinase K at different storage conditions, which resulted in minor variations in proteolytic patterns between experiments.

    Concentration Assay:

    Article Title: Rapid, high-throughput detection of PrPSc by 96-well immunoassay
    Article Snippet: Brain tissue was collected from Syrian golden hamsters infected with either the hyper (HY) or drowsy (DY) strains of transmissible mink encephalopathy (TME) agent at terminal disease or uninfected control hamsters. .. Brain tissue was homogenized to 10% w/v in Dulbecco's phosphate buffered saline (DPBS; Mediatech, Herndon, VA) and was incubated with an equal volume of either proteinase K (final concentration 4 U/ml PK; Roche Diagnostics GmbH, Mannheim, Germany) at 37°C for 1 hour with constant agitation, or diluted in DPBS for undigested controls. .. Following incubation, the samples were 2-fold serially diluted in DPBS.

    Article Title: Degradation of RNA during lysis of Escherichia coli cells in agarose plugs breaks the chromosome
    Article Snippet: The pH of the solution was adjusted by addition of 1/10th volume of 1M Tris HCl (pH 7.4), and the stock was diluted to a final concentration of 2 mg/ml and stored at –20°C. .. Proteinase K was from Roche Applied Science.

    Article Title: Agent strain variation in human prion disease: insights from a molecular and pathological review of the National Institutes of Health series of experimentally transmitted disease
    Article Snippet: Preparation of samples including PrPTSE purification, western blotting and PrPTSE typing were performed according to established methods ( , ; , ). .. In particular, all samples were homogenized in lysis buffer plus [100 mmol/l NaCl, 10 mmol/l EDTA, 0.5% (v/v) Nonidet P 40, 0.5% (w/v) sodium deoxycholate, 100 mmol/l Tris–HCl, pH 6.9] and digested with proteinase K (Roche Diagnostics, specific activity by certificate of analysis: 47.9 U/mg) at a final concentration of 5 U/ml. .. Purified PrPTSE was obtained from about 400 mg of tissue from the cerebral cortex, which was subjected to three cycles of sarkosyl extraction and differential centrifugation to yield the P3 pellet.

    Article Title: Acquired transmissibility of sheep-passaged L-type bovine spongiform encephalopathy prion to wild-type mice
    Article Snippet: The tissues (200 ± 10 mg) were homogenized at 20% concentration (w/v) in a buffer containing 100 mM NaCl and 50 mM Tris–HCl (pH 7.6). .. The sample was then digested with 40 μg/mL proteinase K (PK; Roche Diagnostics, Basel, Switzerland) and the digestion was terminated using 2 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (Pefabloc; Roche Diagnostics).

    Article Title: Lesion of the Olfactory Epithelium Accelerates Prion Neuroinvasion and Disease Onset when Prion Replication Is Restricted to Neurons
    Article Snippet: The protein concentration in tissue homogenates was determined using the micro-BCA assay (Pierce Protein Research, Rockford, IL). .. For detection of PrPSc in brain and olfactory bulb homogenates, protein (100 to 300 μg) from tissues of clinically ill rodents was digested with proteinase K (Roche Diagnostics Corporation, Indianapolis, IN) at 10 μg/ml at a protein sample concentration of 1 mg/ml and incubated at 37°C for one hour with constant agitation followed by the addition of 1 mM PefaBloc (Roche Diagnostics Corporation, Indianapolis, IN). .. To enrich for PrPSc in tissue from rodents during preclinical disease, tissue ( < 10 mg to 200 mg) was extracted with detergent (10% [wt. vol.]

    CTG Assay:

    Article Title: Prion Pathogenesis in the Absence of NLRP3/ASC Inflammasomes
    Article Snippet: Tissue biopsies were digested in 10 mM Tris pH 9, 50 mM KCl, 0.5% Tween20, 0.5% NP40, 0.1 mg/mL proteinase K (Roche) and 1 µL of crude digest was employed as template for a PCR reaction using the Red Taq ReadyMix PCR reaction Mix (Sigma) on a GeneAmp PCR System 9700 (Applied Biosystems). .. For the Nlrp3 tm1Ts c allele, 0.33 μM of each of the following primers were used (5’→3’): AAG TCG TGC TGC TTC ATG T, TCA AGC TAA GAG AAC TTT CTG and ACA CTC GTC ATC TTC AGC A.

    Lysis:

    Article Title: Agent strain variation in human prion disease: insights from a molecular and pathological review of the National Institutes of Health series of experimentally transmitted disease
    Article Snippet: Preparation of samples including PrPTSE purification, western blotting and PrPTSE typing were performed according to established methods ( , ; , ). .. In particular, all samples were homogenized in lysis buffer plus [100 mmol/l NaCl, 10 mmol/l EDTA, 0.5% (v/v) Nonidet P 40, 0.5% (w/v) sodium deoxycholate, 100 mmol/l Tris–HCl, pH 6.9] and digested with proteinase K (Roche Diagnostics, specific activity by certificate of analysis: 47.9 U/mg) at a final concentration of 5 U/ml. .. Purified PrPTSE was obtained from about 400 mg of tissue from the cerebral cortex, which was subjected to three cycles of sarkosyl extraction and differential centrifugation to yield the P3 pellet.

    Article Title: Lesion of the Olfactory Epithelium Accelerates Prion Neuroinvasion and Disease Onset when Prion Replication Is Restricted to Neurons
    Article Snippet: Frozen brain, olfactory bulb and nasal turbinate were homogenized in lysis buffer (i.e., 10 mM Tris-HCL, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% sodium deoxycholate and 0.5% ipegal) containing 1X complete protease inhibitor (Roche Diagnostics, Indianapolis, IN) to approximately 10% (weight per volume). .. For detection of PrPSc in brain and olfactory bulb homogenates, protein (100 to 300 μg) from tissues of clinically ill rodents was digested with proteinase K (Roche Diagnostics Corporation, Indianapolis, IN) at 10 μg/ml at a protein sample concentration of 1 mg/ml and incubated at 37°C for one hour with constant agitation followed by the addition of 1 mM PefaBloc (Roche Diagnostics Corporation, Indianapolis, IN).

    Article Title: Transglutaminase-mediated Intramolecular Cross-linking of Membrane-bound ?-Synuclein Promotes Amyloid Formation in Lewy Bodies
    Article Snippet: Total brain tissue proteins were purified after TriPure lysis and merging TriPure-soluble and insoluble fractions. .. Sample proteins were supplemented with ∼ 0.1 pmol of GGEL (Sigma) as internal (light) standard, subjected to limited acid hydrolysis (0.5 n trifluoroacetic acid) at 95 °C for 16 h), dried, and then digested with proteinase K (Roche Applied Science, 20 μg/ml in 10 m m HEPES-Na, pH 8.0, and 1 m m CaCl2 ) in water containing 20% H2 18 O (Aldrich) at 56 °C overnight.

    Gradient Centrifugation:

    Article Title: Proteomic Characterization of Pseudorabies Virus Extracellular Virions
    Article Snippet: The viral pellet was resuspended in 1 ml of MNT buffer (30 mM morpholineethanesulfonic acid [MES], 10 mM NaCl, and 20 mM Tris-HCl [pH 7.4]), layered over a 10% Ficoll 400 (Sigma-Aldrich) cushion, and centrifuged at 116,000 × g for 2 h at 4°C in a Beckman SW41 Ti rotor. .. For protease treatment of virions, virions were resuspended in 1 ml of MNT buffer containing 10 μg/ml proteinase K (Roche, Mannheim, Germany) for 45 min at room temperature and subsequently treated with 2 mM phenylmethylsulfonyl fluoride (PMSF; Roche) prior to density gradient centrifugation ( ). .. The resulting pellet was washed by resuspension in MNT buffer and concentrated by centrifugation at 116,000 × g for 30 min at 4°C.

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  • 99
    Roche proteinase k
    RCA can detect rSDS-PrP Sc  oligomers at early stages of the disease.  a  Fifty microliters of 10 % hamster brain homogenates (NBH or infected with the 263 K prion strain) were diluted in 300 μL of PBS/2 % Sarkosyl, incubated with 1.5 mM MR100 at room temperature for 1 h and then centrifuged at 8000 g for 5 min. Supernatants (S) were collected and 30 μL of each supernatant were mixed with an equivalent volume of 2X loading buffer. Pellets (P) were resuspended in 30 μL PBS/2 % Sarcosyl, and mixed with 30 μL of 2X loading buffer. Thirty microliters of each sample were loaded on 12 % Bis-tris gels (Criterion, Biorad) and western blotting was carried out with the SAF mix according to standard procedures [  16 ]. Molecular masses (20–75 kDa) are indicated on the left side of the panels.  b  Hamster brain tissues were collected at various days post-infection (d.p.i.), as indicated, and freshly homogenized tissues were processed according to the RCA protocol using MR100 and analyzed by immunoblotting as described above.  c  To compare the RCA and the PK test, the same hamster brain homogenates (at 109, 130 and 148 d.p.i.) were incubated with MR100 at room temperature for 1 h, then digested with 20 μg/mL of proteinase K and processed as described in the legend to Fig.   4a . The asterisk in  b  and  c  indicates the position of oligomer traces
    Proteinase K, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 398 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    RCA can detect rSDS-PrP Sc  oligomers at early stages of the disease.  a  Fifty microliters of 10 % hamster brain homogenates (NBH or infected with the 263 K prion strain) were diluted in 300 μL of PBS/2 % Sarkosyl, incubated with 1.5 mM MR100 at room temperature for 1 h and then centrifuged at 8000 g for 5 min. Supernatants (S) were collected and 30 μL of each supernatant were mixed with an equivalent volume of 2X loading buffer. Pellets (P) were resuspended in 30 μL PBS/2 % Sarcosyl, and mixed with 30 μL of 2X loading buffer. Thirty microliters of each sample were loaded on 12 % Bis-tris gels (Criterion, Biorad) and western blotting was carried out with the SAF mix according to standard procedures [  16 ]. Molecular masses (20–75 kDa) are indicated on the left side of the panels.  b  Hamster brain tissues were collected at various days post-infection (d.p.i.), as indicated, and freshly homogenized tissues were processed according to the RCA protocol using MR100 and analyzed by immunoblotting as described above.  c  To compare the RCA and the PK test, the same hamster brain homogenates (at 109, 130 and 148 d.p.i.) were incubated with MR100 at room temperature for 1 h, then digested with 20 μg/mL of proteinase K and processed as described in the legend to Fig.   4a . The asterisk in  b  and  c  indicates the position of oligomer traces

    Journal: Molecular Neurodegeneration

    Article Title: A Fluorescent Oligothiophene-Bis-Triazine ligand interacts with PrP fibrils and detects SDS-resistant oligomers in human prion diseases

    doi: 10.1186/s13024-016-0074-7

    Figure Lengend Snippet: RCA can detect rSDS-PrP Sc oligomers at early stages of the disease. a Fifty microliters of 10 % hamster brain homogenates (NBH or infected with the 263 K prion strain) were diluted in 300 μL of PBS/2 % Sarkosyl, incubated with 1.5 mM MR100 at room temperature for 1 h and then centrifuged at 8000 g for 5 min. Supernatants (S) were collected and 30 μL of each supernatant were mixed with an equivalent volume of 2X loading buffer. Pellets (P) were resuspended in 30 μL PBS/2 % Sarcosyl, and mixed with 30 μL of 2X loading buffer. Thirty microliters of each sample were loaded on 12 % Bis-tris gels (Criterion, Biorad) and western blotting was carried out with the SAF mix according to standard procedures [ 16 ]. Molecular masses (20–75 kDa) are indicated on the left side of the panels. b Hamster brain tissues were collected at various days post-infection (d.p.i.), as indicated, and freshly homogenized tissues were processed according to the RCA protocol using MR100 and analyzed by immunoblotting as described above. c To compare the RCA and the PK test, the same hamster brain homogenates (at 109, 130 and 148 d.p.i.) were incubated with MR100 at room temperature for 1 h, then digested with 20 μg/mL of proteinase K and processed as described in the legend to Fig.  4a . The asterisk in b and c indicates the position of oligomer traces

    Article Snippet: Pefabloc and proteinase K were purchased from Roche Diagnostics (Mannheim, Germany).

    Techniques: Infection, Incubation, Western Blot

    A MR100-based assay can differentiate between prion-infected and normal brain homogenates without proteinase K digestion.  a  Schematic description of the RCA protocol to test brain homogenates without PK digestion. Brain tissues were freshly homogenized in microbead-containing tubes. Normal brain homogenates (NBH) or prion-infected brain homogenates (IBH) were incubated with MR100 for 1 h, at room temperature, leading to a precipitation of PrP isoforms. After a short centrifugation step, the pellet with MR100 (orange tube) concentrates PrP isoforms, whereas no pellet is detectable with DMSO.  b  Comparison of DMSO, P30 and MR100 precipitation capabilities using the RCA protocol. Fifty μL of 10 % 22 L infected brain homogenates were diluted in 300 μL of PBS/2 % sarcosyl and incubated using either 1.5 mM of P30 or MR100 or an equivalent volume of the solvent alone (DMSO), at room temperature for 1 h. Then, samples were centrifuged at 8000 g for 5 min. Supernatants (S) were collected and 30 μL of each supernatant was mixed with an equivalent volume of 2X loading buffer. Pellets (P) were resuspended in 30 μL PBS/2 % Sarcosyl, and mixed with an equal volume of 2X loading buffer. Thirty microliters of each sample were loaded on 12 % Bis-Tris gels (Criterion, Biorad) and immunoblotting was carried out with the SAF mix according to standard procedures [  16 ]. The samples (S/P) were analyzed by western blotting using SAF mix anti-PrP antibodies.  c  Comparison of infected versus non-infected brain homogenates processed with the RCA protocol. Fifty microliters of 10 % freshly homogenized brain tissues from normal (NBH) or 22 L prion-infected (IBH) mice were processed according to the RCA protocol described in A and B. Thirty microliters of supernatant (S) or pellet (P) were loaded on 12 % Bis-Tris gels (Criterion, Biorad) and immunoblotting was carried out with the SAF mix as described above. Molecular masses (20–75 kDa) are indicated on the left side of the panels

    Journal: Molecular Neurodegeneration

    Article Title: A Fluorescent Oligothiophene-Bis-Triazine ligand interacts with PrP fibrils and detects SDS-resistant oligomers in human prion diseases

    doi: 10.1186/s13024-016-0074-7

    Figure Lengend Snippet: A MR100-based assay can differentiate between prion-infected and normal brain homogenates without proteinase K digestion. a Schematic description of the RCA protocol to test brain homogenates without PK digestion. Brain tissues were freshly homogenized in microbead-containing tubes. Normal brain homogenates (NBH) or prion-infected brain homogenates (IBH) were incubated with MR100 for 1 h, at room temperature, leading to a precipitation of PrP isoforms. After a short centrifugation step, the pellet with MR100 (orange tube) concentrates PrP isoforms, whereas no pellet is detectable with DMSO. b Comparison of DMSO, P30 and MR100 precipitation capabilities using the RCA protocol. Fifty μL of 10 % 22 L infected brain homogenates were diluted in 300 μL of PBS/2 % sarcosyl and incubated using either 1.5 mM of P30 or MR100 or an equivalent volume of the solvent alone (DMSO), at room temperature for 1 h. Then, samples were centrifuged at 8000 g for 5 min. Supernatants (S) were collected and 30 μL of each supernatant was mixed with an equivalent volume of 2X loading buffer. Pellets (P) were resuspended in 30 μL PBS/2 % Sarcosyl, and mixed with an equal volume of 2X loading buffer. Thirty microliters of each sample were loaded on 12 % Bis-Tris gels (Criterion, Biorad) and immunoblotting was carried out with the SAF mix according to standard procedures [ 16 ]. The samples (S/P) were analyzed by western blotting using SAF mix anti-PrP antibodies. c Comparison of infected versus non-infected brain homogenates processed with the RCA protocol. Fifty microliters of 10 % freshly homogenized brain tissues from normal (NBH) or 22 L prion-infected (IBH) mice were processed according to the RCA protocol described in A and B. Thirty microliters of supernatant (S) or pellet (P) were loaded on 12 % Bis-Tris gels (Criterion, Biorad) and immunoblotting was carried out with the SAF mix as described above. Molecular masses (20–75 kDa) are indicated on the left side of the panels

    Article Snippet: Pefabloc and proteinase K were purchased from Roche Diagnostics (Mannheim, Germany).

    Techniques: Infection, Incubation, Centrifugation, Western Blot, Mouse Assay

    rSDS-PrP Sc  oligomers are detected in patients with new variant CJD (vCJD) when tested with RCA.  a-b  Frozen, homogenized brain samples from two patients with vCJD, one patient with sCJD (codon 129 M/M genotype) (positive control) and one healthy control (NBH) were from NIBSC. Each sample was identified by the number attributed by the NIBSC. The RCA assay was carried out as before (see legend to Fig.   5 ) but adapted to human samples: 50 μL of 10 % brain homogenates in 100 μL PBS/2 % Sarkosyl were incubated with 2 mM MR100 for 2 h. Before centrifugation, 30 μL was collected and mixed with 30 μL of 2X loading buffer for immunoblotting analysis ( a ). The rest of the sample was centrifuged at 11,000 g for 5 min, and supernatants (S) and pellets (P) were immunoblotted with the SAFmix ( b ) [  16 ].  c-d  For comparison, the same brain homogenates (50 μL of 10 % brain homogenates in 100 μL PBS/2 % Sarkosyl) were processed with the classical proteinase K digestion assay without MR100. Samples were analyzed before ( c ), and after proteinase K digestion (125 μg/mL PK at 37 °C for 1 h) ( d ). The reaction was stopped by addition of a protease inhibitor cocktail, before analysis of rPrP Sc  by western blotting with the SAF mix. Molecular masses (20–75 kDa) are on the left side of the panels

    Journal: Molecular Neurodegeneration

    Article Title: A Fluorescent Oligothiophene-Bis-Triazine ligand interacts with PrP fibrils and detects SDS-resistant oligomers in human prion diseases

    doi: 10.1186/s13024-016-0074-7

    Figure Lengend Snippet: rSDS-PrP Sc oligomers are detected in patients with new variant CJD (vCJD) when tested with RCA. a-b Frozen, homogenized brain samples from two patients with vCJD, one patient with sCJD (codon 129 M/M genotype) (positive control) and one healthy control (NBH) were from NIBSC. Each sample was identified by the number attributed by the NIBSC. The RCA assay was carried out as before (see legend to Fig.  5 ) but adapted to human samples: 50 μL of 10 % brain homogenates in 100 μL PBS/2 % Sarkosyl were incubated with 2 mM MR100 for 2 h. Before centrifugation, 30 μL was collected and mixed with 30 μL of 2X loading buffer for immunoblotting analysis ( a ). The rest of the sample was centrifuged at 11,000 g for 5 min, and supernatants (S) and pellets (P) were immunoblotted with the SAFmix ( b ) [ 16 ]. c-d For comparison, the same brain homogenates (50 μL of 10 % brain homogenates in 100 μL PBS/2 % Sarkosyl) were processed with the classical proteinase K digestion assay without MR100. Samples were analyzed before ( c ), and after proteinase K digestion (125 μg/mL PK at 37 °C for 1 h) ( d ). The reaction was stopped by addition of a protease inhibitor cocktail, before analysis of rPrP Sc by western blotting with the SAF mix. Molecular masses (20–75 kDa) are on the left side of the panels

    Article Snippet: Pefabloc and proteinase K were purchased from Roche Diagnostics (Mannheim, Germany).

    Techniques: Variant Assay, Positive Control, Incubation, Centrifugation, Protease Inhibitor, Western Blot

    MR100 has a stronger PrP Sc  oligomer-inducing activity in prion-infected N2a58/22 L cells than P30 and A6.  a  Effect of the newly synthesized MR1, MR2 and MR100 compounds in prion-infected N2a58/22 L cells. Cells were left untreated (CTR) or incubated with 20 μM of A6 (positive control), MR1 and MR2 (synthesis intermediates), MR100, or 20 μL DMSO (DM) for 4 days. Protein lysates were analyzed by immunoblotting with the SAF mix (a mixture of the anti-PrP SAF60, SAF69 and SAF70 monoclonal antibodies) after proteinase K (PK) digestion.  b  Comparison of the oligomer-inducing activity of P30, A6 and MR100. Prion-infected N2a58/22 L cells were incubated with 0.5, 1 or 2.5 μM of each compound, or 20 μL DMSO (DM) for 4 days. Protein lysates were then analyzed by immunoblotting with the SAF mix after PK digestion.  c  MR100 dose-response curve in prion-infected N2a58/22 L cells. Successive dilutions of MR100 in DMSO were used to obtain final concentrations ranging from 10 -12 M (1pM) to 10 -5 M (10 μM). Cells were incubated for 4 days and at confluence they were lysed. Protein lysates were analyzed by immunoblotting with the SAF mix after PK digestion according to the previously described protocol [  16 ,   30 ]. Loading control was performed with antibodies against glyceraldehyde-3-P dehydrogenase (G3PDH) and before proteinase K digestion. Molecular masses (20–50 kDa) are indicated on the left side of the panels

    Journal: Molecular Neurodegeneration

    Article Title: A Fluorescent Oligothiophene-Bis-Triazine ligand interacts with PrP fibrils and detects SDS-resistant oligomers in human prion diseases

    doi: 10.1186/s13024-016-0074-7

    Figure Lengend Snippet: MR100 has a stronger PrP Sc oligomer-inducing activity in prion-infected N2a58/22 L cells than P30 and A6. a Effect of the newly synthesized MR1, MR2 and MR100 compounds in prion-infected N2a58/22 L cells. Cells were left untreated (CTR) or incubated with 20 μM of A6 (positive control), MR1 and MR2 (synthesis intermediates), MR100, or 20 μL DMSO (DM) for 4 days. Protein lysates were analyzed by immunoblotting with the SAF mix (a mixture of the anti-PrP SAF60, SAF69 and SAF70 monoclonal antibodies) after proteinase K (PK) digestion. b Comparison of the oligomer-inducing activity of P30, A6 and MR100. Prion-infected N2a58/22 L cells were incubated with 0.5, 1 or 2.5 μM of each compound, or 20 μL DMSO (DM) for 4 days. Protein lysates were then analyzed by immunoblotting with the SAF mix after PK digestion. c MR100 dose-response curve in prion-infected N2a58/22 L cells. Successive dilutions of MR100 in DMSO were used to obtain final concentrations ranging from 10 -12 M (1pM) to 10 -5 M (10 μM). Cells were incubated for 4 days and at confluence they were lysed. Protein lysates were analyzed by immunoblotting with the SAF mix after PK digestion according to the previously described protocol [ 16 , 30 ]. Loading control was performed with antibodies against glyceraldehyde-3-P dehydrogenase (G3PDH) and before proteinase K digestion. Molecular masses (20–50 kDa) are indicated on the left side of the panels

    Article Snippet: Pefabloc and proteinase K were purchased from Roche Diagnostics (Mannheim, Germany).

    Techniques: Activity Assay, Infection, Synthesized, Incubation, Positive Control

    MR100 did not induce SDS resistant PrP C  oligomers.  a  Parental non-infected cells, N2a58, were left untreated (CTR) or incubated with 20 μM of MR100 or 20 μL DMSO (DM) for 4 days. Protein lysates were analyzed by immunoblotting with the SAF mix before or after PK digestion.  b  N2a58 cell lines were left untreated (CTR) or incubated with various concentration of MR100 from 5 to 40 μM or 40 μL of DMSO (DM) for 4 days. Protein lysates were analyzed by immunoblotting with the SAF 32 before or after PK digestion. Loading control was performed with antibodies against β actin and before proteinase K digestion.  c  Schematic representation of the protocols used to test if PrP C  isoforms are part of rSDS-oligomers (Left panel). First step, N2a58/22 L lysates were incubated with 20 μM of MR100 or with proteinase K to eliminate PrP C , then in the second step, MR100-exposed lysates were digested with proteinase K, while proteinase K digested samples were incubated with MR100. PrP Sc  species were then analyzed by western blotting. Western blot analysis of the samples processed according to the two different protocols using the SAFmix of anti-PrP antibodies (Right panel). CTR, untreated samples, digested by proteinase K; DM, DMSO. Molecular masses (20–50 kDa) are indicated on the left side of the panels

    Journal: Molecular Neurodegeneration

    Article Title: A Fluorescent Oligothiophene-Bis-Triazine ligand interacts with PrP fibrils and detects SDS-resistant oligomers in human prion diseases

    doi: 10.1186/s13024-016-0074-7

    Figure Lengend Snippet: MR100 did not induce SDS resistant PrP C oligomers. a Parental non-infected cells, N2a58, were left untreated (CTR) or incubated with 20 μM of MR100 or 20 μL DMSO (DM) for 4 days. Protein lysates were analyzed by immunoblotting with the SAF mix before or after PK digestion. b N2a58 cell lines were left untreated (CTR) or incubated with various concentration of MR100 from 5 to 40 μM or 40 μL of DMSO (DM) for 4 days. Protein lysates were analyzed by immunoblotting with the SAF 32 before or after PK digestion. Loading control was performed with antibodies against β actin and before proteinase K digestion. c Schematic representation of the protocols used to test if PrP C isoforms are part of rSDS-oligomers (Left panel). First step, N2a58/22 L lysates were incubated with 20 μM of MR100 or with proteinase K to eliminate PrP C , then in the second step, MR100-exposed lysates were digested with proteinase K, while proteinase K digested samples were incubated with MR100. PrP Sc species were then analyzed by western blotting. Western blot analysis of the samples processed according to the two different protocols using the SAFmix of anti-PrP antibodies (Right panel). CTR, untreated samples, digested by proteinase K; DM, DMSO. Molecular masses (20–50 kDa) are indicated on the left side of the panels

    Article Snippet: Pefabloc and proteinase K were purchased from Roche Diagnostics (Mannheim, Germany).

    Techniques: Infection, Incubation, Concentration Assay, Western Blot

    MR100 shows oligomer-inducing activity in brain homogenates from prion-infected rodents.  a  MR100 oligomer-inducing activity was tested using freshly homogenized rodent brain tissues infected with the 22 L (mice) or the 263 K prion strain (hamsters). Fifty μL of 10 % mouse or hamster brain homogenates were diluted in 300 μL PBS/2 % Sarkosyl, incubated with 1.5 mM MR100 (corresponding to 150 μL of 5 mM MR100) at room temperature for 1 h or with 150 μL of DMSO as control, and then digested with 20 μg/ml PK at a ratio of 1:50 (PK/proteins). PK digestion was stopped by addition of a cocktail of protease inhibitors (Complete), before analysis of rPrP Sc  by western blotting with the SAF mix according to the previously described protocol [  16 ]. CTR: untreated 22 L- or 263 K-infected brain homogenates (negative control); DM: 22 L- or 263 K-infected brain homogenates incubated with DMSO.  b  Comparison of P30 and MR100 oligomer-inducing activity on the 22 L prion strain, before and after proteinase K digestion. Fifty μL of 10 % 22 L-infected brain homogenates were diluted in 350 μL PBS/2 % Sarkosyl, incubated with 1 mM MR100 or P30 (corresponding to 100 μL of 5 mM MR100 or P30), at room temperature for 1 h. Then, aliquots of 30 μL were taken before addition of proteinase K, to perform western blot (PK-) probed with SAF mix, but also with anti-β-actin antibodies as loading controls. The rest of the sample was then digested with 20 μg/ml PK at a ratio of 1:50 (PK/proteins) (PK+). The reaction was stopped by addition of the protease inhibitor cocktail, before analysis of rPrP Sc  by western blotting with the SAF mix as in A. DM: 22 L-infected brain homogenates incubated with 100 μL DMSO. Molecular masses (20–50 kDa) are indicated on the left side of the panels

    Journal: Molecular Neurodegeneration

    Article Title: A Fluorescent Oligothiophene-Bis-Triazine ligand interacts with PrP fibrils and detects SDS-resistant oligomers in human prion diseases

    doi: 10.1186/s13024-016-0074-7

    Figure Lengend Snippet: MR100 shows oligomer-inducing activity in brain homogenates from prion-infected rodents. a MR100 oligomer-inducing activity was tested using freshly homogenized rodent brain tissues infected with the 22 L (mice) or the 263 K prion strain (hamsters). Fifty μL of 10 % mouse or hamster brain homogenates were diluted in 300 μL PBS/2 % Sarkosyl, incubated with 1.5 mM MR100 (corresponding to 150 μL of 5 mM MR100) at room temperature for 1 h or with 150 μL of DMSO as control, and then digested with 20 μg/ml PK at a ratio of 1:50 (PK/proteins). PK digestion was stopped by addition of a cocktail of protease inhibitors (Complete), before analysis of rPrP Sc by western blotting with the SAF mix according to the previously described protocol [ 16 ]. CTR: untreated 22 L- or 263 K-infected brain homogenates (negative control); DM: 22 L- or 263 K-infected brain homogenates incubated with DMSO. b Comparison of P30 and MR100 oligomer-inducing activity on the 22 L prion strain, before and after proteinase K digestion. Fifty μL of 10 % 22 L-infected brain homogenates were diluted in 350 μL PBS/2 % Sarkosyl, incubated with 1 mM MR100 or P30 (corresponding to 100 μL of 5 mM MR100 or P30), at room temperature for 1 h. Then, aliquots of 30 μL were taken before addition of proteinase K, to perform western blot (PK-) probed with SAF mix, but also with anti-β-actin antibodies as loading controls. The rest of the sample was then digested with 20 μg/ml PK at a ratio of 1:50 (PK/proteins) (PK+). The reaction was stopped by addition of the protease inhibitor cocktail, before analysis of rPrP Sc by western blotting with the SAF mix as in A. DM: 22 L-infected brain homogenates incubated with 100 μL DMSO. Molecular masses (20–50 kDa) are indicated on the left side of the panels

    Article Snippet: Pefabloc and proteinase K were purchased from Roche Diagnostics (Mannheim, Germany).

    Techniques: Activity Assay, Infection, Mouse Assay, Incubation, Western Blot, Negative Control, Protease Inhibitor

    Effect of accessory cofactors on the propagation of ΔPBD PrP Sc  molecules. In vitro -generated ΔC-PBD, ΔN-PBD, and ΔΔ-PBD PrP Sc  molecules were propagated by sPMCA with autologous PrP C  prepared from Chinese hamster ovary (CHO) cells. One set of reactions was supplemented with  Prnp 0/0  mouse brain homogenate (+cofactor), while a second set received only buffer (−cofactor). One sample of each reaction was not subjected to protease digestion (−PK), while all others were digested with 25 µg/mL proteinase K. –PK reactions for ΔC-PBD and ΔΔ-PBD were loaded with ¼ volume. PrP was detected by immunoblot (anti-PrP 27/33).

    Journal: PLoS Pathogens

    Article Title: Dissociation of Infectivity from Seeding Ability in Prions with Alternate Docking Mechanism

    doi: 10.1371/journal.ppat.1002128

    Figure Lengend Snippet: Effect of accessory cofactors on the propagation of ΔPBD PrP Sc molecules. In vitro -generated ΔC-PBD, ΔN-PBD, and ΔΔ-PBD PrP Sc molecules were propagated by sPMCA with autologous PrP C prepared from Chinese hamster ovary (CHO) cells. One set of reactions was supplemented with Prnp 0/0 mouse brain homogenate (+cofactor), while a second set received only buffer (−cofactor). One sample of each reaction was not subjected to protease digestion (−PK), while all others were digested with 25 µg/mL proteinase K. –PK reactions for ΔC-PBD and ΔΔ-PBD were loaded with ¼ volume. PrP was detected by immunoblot (anti-PrP 27/33).

    Article Snippet: Mouse PrPSc was detected by digestion with 25 µg/mL proteinase K (Roche, Indianapolis, IN) for 30 min. at 37°C and 750 r.p.m. shaking, polyacrylamide gel electrophoresis (PAGE), transfer to polyvinylidene fluoride (PVDF), and Western detection with anti-PrP and horseradish peroxidase-conjugated anti-mouse sheep IgG (GE Healthcare, Piscataway, NJ).

    Techniques: In Vitro, Generated

    Biochemical and neuropathological analysis of mice inoculated with  in vitro -generated PrP Sc  molecules. Brains were dissected from wild-type mice showing terminal scrapie signs (PrP Sc  and ΔC-PBD PrP Sc  inocula) or similarly aged mice not displaying scrapie signs (mock-propagated, ΔN-PBD PrP Sc , and ΔΔ-PBD PrP Sc  inocula). ( A ) Equivalent amounts of 10% brain homogenate were treated with buffer (−PK) or 25 µg/mL proteinase K (+PK to show PrP Sc ) and detected by anti-PrP (6D11) immunoblot. A greater exposure of the same immunoblot is displayed below, to illustrate samples containing low amounts of PrP Sc . ( B ) Neuropathology of cerebellum and hippocampus. Brain sections were stained with hematoxylin and eosin (H  E). The black bar denotes 100 µm.

    Journal: PLoS Pathogens

    Article Title: Dissociation of Infectivity from Seeding Ability in Prions with Alternate Docking Mechanism

    doi: 10.1371/journal.ppat.1002128

    Figure Lengend Snippet: Biochemical and neuropathological analysis of mice inoculated with in vitro -generated PrP Sc molecules. Brains were dissected from wild-type mice showing terminal scrapie signs (PrP Sc and ΔC-PBD PrP Sc inocula) or similarly aged mice not displaying scrapie signs (mock-propagated, ΔN-PBD PrP Sc , and ΔΔ-PBD PrP Sc inocula). ( A ) Equivalent amounts of 10% brain homogenate were treated with buffer (−PK) or 25 µg/mL proteinase K (+PK to show PrP Sc ) and detected by anti-PrP (6D11) immunoblot. A greater exposure of the same immunoblot is displayed below, to illustrate samples containing low amounts of PrP Sc . ( B ) Neuropathology of cerebellum and hippocampus. Brain sections were stained with hematoxylin and eosin (H E). The black bar denotes 100 µm.

    Article Snippet: Mouse PrPSc was detected by digestion with 25 µg/mL proteinase K (Roche, Indianapolis, IN) for 30 min. at 37°C and 750 r.p.m. shaking, polyacrylamide gel electrophoresis (PAGE), transfer to polyvinylidene fluoride (PVDF), and Western detection with anti-PrP and horseradish peroxidase-conjugated anti-mouse sheep IgG (GE Healthcare, Piscataway, NJ).

    Techniques: Mouse Assay, In Vitro, Generated, Staining

    Interaction of ΔC-PBD PrP Sc  with mutant PrP molecules. ( A ) Binding of ΔC-PBD PrP Sc  to PrP. ΔC-PBD PrP Sc  was incubated with wild-type or polybasic mutant (ΔC-PBD, ΔN-PBD) myc-tagged PrP. Bound PrP Sc  was captured with 9E10 anti-myc antibody on magnetic protein A Dynabeads, and detected by 25 µg/mL proteinase K digestion and anti-PrP (27/33) immunoblot. ( B ) Propagation of ΔC-PBD PrP Sc . ΔC-PBD PrP Sc  was propagated by sPMCA with polybasic deletion mutant PrP C  prepared from Chinese hamster ovary (CHO) cells. Reactions were supplemented with  Prnp 0/0  mouse brain homogenate. One sample of each reaction was not subjected to protease digestion (−PK), loading ¼ of volume. All others were subjected to limited proteolysis by 25 µg/mL proteinase K digestion. PrP was detected by immunoblot (anti-PrP 27/33).

    Journal: PLoS Pathogens

    Article Title: Dissociation of Infectivity from Seeding Ability in Prions with Alternate Docking Mechanism

    doi: 10.1371/journal.ppat.1002128

    Figure Lengend Snippet: Interaction of ΔC-PBD PrP Sc with mutant PrP molecules. ( A ) Binding of ΔC-PBD PrP Sc to PrP. ΔC-PBD PrP Sc was incubated with wild-type or polybasic mutant (ΔC-PBD, ΔN-PBD) myc-tagged PrP. Bound PrP Sc was captured with 9E10 anti-myc antibody on magnetic protein A Dynabeads, and detected by 25 µg/mL proteinase K digestion and anti-PrP (27/33) immunoblot. ( B ) Propagation of ΔC-PBD PrP Sc . ΔC-PBD PrP Sc was propagated by sPMCA with polybasic deletion mutant PrP C prepared from Chinese hamster ovary (CHO) cells. Reactions were supplemented with Prnp 0/0 mouse brain homogenate. One sample of each reaction was not subjected to protease digestion (−PK), loading ¼ of volume. All others were subjected to limited proteolysis by 25 µg/mL proteinase K digestion. PrP was detected by immunoblot (anti-PrP 27/33).

    Article Snippet: Mouse PrPSc was detected by digestion with 25 µg/mL proteinase K (Roche, Indianapolis, IN) for 30 min. at 37°C and 750 r.p.m. shaking, polyacrylamide gel electrophoresis (PAGE), transfer to polyvinylidene fluoride (PVDF), and Western detection with anti-PrP and horseradish peroxidase-conjugated anti-mouse sheep IgG (GE Healthcare, Piscataway, NJ).

    Techniques: Mutagenesis, Binding Assay, Incubation

    ΔPBD PrP Sc  molecules seeding wild-type brain homogenate sPMCA reactions. In vitro -generated ΔC-PBD, ΔN-PBD, and ΔΔ-PBD PrP Sc  molecules were propagated by sPMCA for three rounds with wild-type mouse brain homogenate, containing wild-type PrP C  substrate. Control reactions seeded with wild-type native RML prions and unseeded reactions were also tested. One sample of each reaction was not subjected to protease digestion (−PK), while all others were digested with 25 µg/mL proteinase K. PrP was detected by immunoblot (anti-PrP 6D11).

    Journal: PLoS Pathogens

    Article Title: Dissociation of Infectivity from Seeding Ability in Prions with Alternate Docking Mechanism

    doi: 10.1371/journal.ppat.1002128

    Figure Lengend Snippet: ΔPBD PrP Sc molecules seeding wild-type brain homogenate sPMCA reactions. In vitro -generated ΔC-PBD, ΔN-PBD, and ΔΔ-PBD PrP Sc molecules were propagated by sPMCA for three rounds with wild-type mouse brain homogenate, containing wild-type PrP C substrate. Control reactions seeded with wild-type native RML prions and unseeded reactions were also tested. One sample of each reaction was not subjected to protease digestion (−PK), while all others were digested with 25 µg/mL proteinase K. PrP was detected by immunoblot (anti-PrP 6D11).

    Article Snippet: Mouse PrPSc was detected by digestion with 25 µg/mL proteinase K (Roche, Indianapolis, IN) for 30 min. at 37°C and 750 r.p.m. shaking, polyacrylamide gel electrophoresis (PAGE), transfer to polyvinylidene fluoride (PVDF), and Western detection with anti-PrP and horseradish peroxidase-conjugated anti-mouse sheep IgG (GE Healthcare, Piscataway, NJ).

    Techniques: In Vitro, Generated

    Interaction of wild-type PrP Sc  with mutant PrP molecules. ( A ) Binding of PrP Sc  to PrP. RML scrapie-infected mouse brain homogenate was incubated with myc-tagged PrP of wild-type sequence or lacking the central (ΔC-PBD: Δ100–109) or N-terminal (ΔN-PBD: Δ23–28) polybasic domain. Bound PrP Sc  was captured with 9E10 anti-myc antibody on magnetic protein A Dynabeads, and detected by 25 µg/mL proteinase K digestion and anti-PrP (6D11) immunoblot. ( B ) Propagation of PrP Sc . RML scrapie-infected mouse brain homogenate was propagated by serial protein misfolding cyclic amplification (sPMCA) for five rounds with wild-type or polybasic deletion mutant PrP C  prepared from Chinese hamster ovary (CHO) cells. Reactions were supplemented with  Prnp 0/0  mouse brain homogenate. In addition to scrapie-seeded reactions, an unseeded reaction was performed with ΔC-PBD PrP substrate. One sample of each reaction was not subjected to protease digestion (−PK). All others were subjected to limited proteolysis with 25 µg/mL proteinase K. PrP was detected by immunoblot (anti-PrP 27/33).

    Journal: PLoS Pathogens

    Article Title: Dissociation of Infectivity from Seeding Ability in Prions with Alternate Docking Mechanism

    doi: 10.1371/journal.ppat.1002128

    Figure Lengend Snippet: Interaction of wild-type PrP Sc with mutant PrP molecules. ( A ) Binding of PrP Sc to PrP. RML scrapie-infected mouse brain homogenate was incubated with myc-tagged PrP of wild-type sequence or lacking the central (ΔC-PBD: Δ100–109) or N-terminal (ΔN-PBD: Δ23–28) polybasic domain. Bound PrP Sc was captured with 9E10 anti-myc antibody on magnetic protein A Dynabeads, and detected by 25 µg/mL proteinase K digestion and anti-PrP (6D11) immunoblot. ( B ) Propagation of PrP Sc . RML scrapie-infected mouse brain homogenate was propagated by serial protein misfolding cyclic amplification (sPMCA) for five rounds with wild-type or polybasic deletion mutant PrP C prepared from Chinese hamster ovary (CHO) cells. Reactions were supplemented with Prnp 0/0 mouse brain homogenate. In addition to scrapie-seeded reactions, an unseeded reaction was performed with ΔC-PBD PrP substrate. One sample of each reaction was not subjected to protease digestion (−PK). All others were subjected to limited proteolysis with 25 µg/mL proteinase K. PrP was detected by immunoblot (anti-PrP 27/33).

    Article Snippet: Mouse PrPSc was detected by digestion with 25 µg/mL proteinase K (Roche, Indianapolis, IN) for 30 min. at 37°C and 750 r.p.m. shaking, polyacrylamide gel electrophoresis (PAGE), transfer to polyvinylidene fluoride (PVDF), and Western detection with anti-PrP and horseradish peroxidase-conjugated anti-mouse sheep IgG (GE Healthcare, Piscataway, NJ).

    Techniques: Mutagenesis, Binding Assay, Infection, Incubation, Sequencing, Protein Misfolding Cyclic Amplification

    PrP CWD  amplification in raw water samples from non-CWD-endemic areas. All samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. Lane 2 shows amplified NBH control. sPMCA failed to amplify any PrP CWD  from five replicate

    Journal:

    Article Title: Detection of protease-resistant cervid prion protein in water from a CWD-endemic area

    doi:

    Figure Lengend Snippet: PrP CWD amplification in raw water samples from non-CWD-endemic areas. All samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. Lane 2 shows amplified NBH control. sPMCA failed to amplify any PrP CWD from five replicate

    Article Snippet: Amplified samples were treated with 50 µg/ml of Proteinase-K (Roche) for 30 min at 45°C and PK was inactivated by the addition of lithium dodecyl sulfate loading buffer (Invitrogen) and heat inactivation for five minutes at 99°C.

    Techniques: Amplification

    Effects of flocculation and alum on PrP CWD  amplification. All samples were digested with Proteinase K except lane 1. (A) PrP CWD  precipitates with flocculant water sample. Lanes 1 and 2 shows amplified NBH control. Lanes 3–6 show amplified samples

    Journal:

    Article Title: Detection of protease-resistant cervid prion protein in water from a CWD-endemic area

    doi:

    Figure Lengend Snippet: Effects of flocculation and alum on PrP CWD amplification. All samples were digested with Proteinase K except lane 1. (A) PrP CWD precipitates with flocculant water sample. Lanes 1 and 2 shows amplified NBH control. Lanes 3–6 show amplified samples

    Article Snippet: Amplified samples were treated with 50 µg/ml of Proteinase-K (Roche) for 30 min at 45°C and PK was inactivated by the addition of lithium dodecyl sulfate loading buffer (Invitrogen) and heat inactivation for five minutes at 99°C.

    Techniques: Flocculation, Amplification

    PrP CWD  detection limit in water. (A) All samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. Lanes 2 through 12 show amplified samples at the indicated starting dilution of CWD-positive brain into water. Lanes 13 and

    Journal:

    Article Title: Detection of protease-resistant cervid prion protein in water from a CWD-endemic area

    doi:

    Figure Lengend Snippet: PrP CWD detection limit in water. (A) All samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. Lanes 2 through 12 show amplified samples at the indicated starting dilution of CWD-positive brain into water. Lanes 13 and

    Article Snippet: Amplified samples were treated with 50 µg/ml of Proteinase-K (Roche) for 30 min at 45°C and PK was inactivated by the addition of lithium dodecyl sulfate loading buffer (Invitrogen) and heat inactivation for five minutes at 99°C.

    Techniques: Amplification

    PrP CWD  detected in raw water samples collected at a time of increased snow-melt runoff. All samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. Lane 2 shows amplified NBH control. Lanes 3–8 show Horsetooth reservoir

    Journal:

    Article Title: Detection of protease-resistant cervid prion protein in water from a CWD-endemic area

    doi:

    Figure Lengend Snippet: PrP CWD detected in raw water samples collected at a time of increased snow-melt runoff. All samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. Lane 2 shows amplified NBH control. Lanes 3–8 show Horsetooth reservoir

    Article Snippet: Amplified samples were treated with 50 µg/ml of Proteinase-K (Roche) for 30 min at 45°C and PK was inactivated by the addition of lithium dodecyl sulfate loading buffer (Invitrogen) and heat inactivation for five minutes at 99°C.

    Techniques: Amplification

    Abrogation of PrP CWD  amplification from 5-22-07 raw PR water samples by PMCA using murine PrP C  substrate or Proteinase K pre-treatment. All western blot samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. Lane 2 shows

    Journal:

    Article Title: Detection of protease-resistant cervid prion protein in water from a CWD-endemic area

    doi:

    Figure Lengend Snippet: Abrogation of PrP CWD amplification from 5-22-07 raw PR water samples by PMCA using murine PrP C substrate or Proteinase K pre-treatment. All western blot samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. Lane 2 shows

    Article Snippet: Amplified samples were treated with 50 µg/ml of Proteinase-K (Roche) for 30 min at 45°C and PK was inactivated by the addition of lithium dodecyl sulfate loading buffer (Invitrogen) and heat inactivation for five minutes at 99°C.

    Techniques: Amplification, Western Blot

    Normal brain homogenate negative controls. All samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. NBH, Normal brain homogenate control. +, 1:100,000 positive amplification control. Molecular weight markers in kilodaltons

    Journal:

    Article Title: Detection of protease-resistant cervid prion protein in water from a CWD-endemic area

    doi:

    Figure Lengend Snippet: Normal brain homogenate negative controls. All samples were digested with Proteinase K except normal brain homogenate (NBH) in lane 1. NBH, Normal brain homogenate control. +, 1:100,000 positive amplification control. Molecular weight markers in kilodaltons

    Article Snippet: Amplified samples were treated with 50 µg/ml of Proteinase-K (Roche) for 30 min at 45°C and PK was inactivated by the addition of lithium dodecyl sulfate loading buffer (Invitrogen) and heat inactivation for five minutes at 99°C.

    Techniques: Amplification, Molecular Weight

    TvTom40-2 is involved in hydrogenosomal protein import. (A) Autoradiograph showing a time-dependent in vitro import of  35 S-Met-labeled TvFdx1-DHFR into hydrogenosomes. (B) Autoradiograph showing the in vitro import of  35 S-Met-labeled TvFdx1-DHFR into hydrogenosomes in either the absence (−) or the presence (+) of MTX, followed by proteinase K (+) treatment. (C) Autoradiograph showing the eluates for the TvTom40-2-HA coIP following the in vitro import of  35 S-Met-labeled TvFdx1-DHFR into hydrogenosomes isolated from a strain expressing both TvTom40-2-HA and Tom36-V5 either in the presence (+) or the absence (−) of MTX. (D) Immunoblot of the same eluates as in panel C using α-HA, α-V5, and α-Sam50 antibodies. coIP, co-immunoprecipitation; DHFR, dihydrofolate reductase; Fdx, ferredoxin; HA, human influenza hemagglutinin; In, input; MTX, methotrexate; PK, proteinase K; Sam, sorting and assembly machinery; TOM, translocase of the outer membrane; TvTom,  T .  vaginalis  TOM.

    Journal: PLoS Biology

    Article Title: Triplet-pore structure of a highly divergent TOM complex of hydrogenosomes in Trichomonas vaginalis

    doi: 10.1371/journal.pbio.3000098

    Figure Lengend Snippet: TvTom40-2 is involved in hydrogenosomal protein import. (A) Autoradiograph showing a time-dependent in vitro import of 35 S-Met-labeled TvFdx1-DHFR into hydrogenosomes. (B) Autoradiograph showing the in vitro import of 35 S-Met-labeled TvFdx1-DHFR into hydrogenosomes in either the absence (−) or the presence (+) of MTX, followed by proteinase K (+) treatment. (C) Autoradiograph showing the eluates for the TvTom40-2-HA coIP following the in vitro import of 35 S-Met-labeled TvFdx1-DHFR into hydrogenosomes isolated from a strain expressing both TvTom40-2-HA and Tom36-V5 either in the presence (+) or the absence (−) of MTX. (D) Immunoblot of the same eluates as in panel C using α-HA, α-V5, and α-Sam50 antibodies. coIP, co-immunoprecipitation; DHFR, dihydrofolate reductase; Fdx, ferredoxin; HA, human influenza hemagglutinin; In, input; MTX, methotrexate; PK, proteinase K; Sam, sorting and assembly machinery; TOM, translocase of the outer membrane; TvTom, T . vaginalis TOM.

    Article Snippet: Isolated hydrogenosomes (protein concentration 1 mg/mL) carrying either HA-tagged or V5-tagged proteins were washed to remove protease inhibitors and incubated for 30 minutes at 37 °C in isolation buffer (225 mM sucrose, 10 mM KH2 PO4 , 20 mM HEPES, 0.5 mM KCl, 5 mM MgCl2 , and 1 mM EDTA [pH 7.2]) supplemented with either 100 μg/mL proteinase K enzyme (Roche Holding AG, Basel, Switzerland) or proteinase K with 0.5% Triton X-100.

    Techniques: Autoradiography, In Vitro, Labeling, Hemagglutination Assay, Co-Immunoprecipitation Assay, Isolation, Expressing, Immunoprecipitation

    Localisation and topology of the TA proteins. (A) Immunoblot analysis of TA proteins in  T .  vaginalis  subcellular fractions using α-V5 and α-αSCS (hydrogenosomal matrix protein) antibodies. Total cell lysates, cytoplasm, hydrogenosomes, hydrogenosomes treated with either proteinase K, or hydrogenosomes treated with proteinase K and Triton X-100 isolated from the strains expressing V5-tagged Tom36, Tom46, Homp38, Homp19, and Tom22-like protein. (B) Double transfectants expressing HA-tagged TvTom40-2 along with one of the V5-tagged proteins, Tom36, Tom46, Homp38, Homp19 or Tom22-like protein were visualised using mouse α-HA/α-mouse Abberior STAR 580 (green) and rabbit α-V5/α-rabbit Abberior STAR 635p (red) antibodies. Scale bar, 1 μm. αSCS, α-subunit of succinyl CoA synthetase; CoA, coenzyme A; Cyt, cytoplasm; H/PK, hydrogenosomes treated with proteinase K; H/TX/PK, hydrogenosomes treated with proteinase K in the presence of Triton X-100; HA, human influenza hemagglutinin; Homp, hydrogenosomal outer membrane protein; Hyd, hydrogenosomes; Lys, lysate; TA, tail-anchored; TOM, translocase of the outer membrane; TvTom,  T .  vaginalis  TOM.

    Journal: PLoS Biology

    Article Title: Triplet-pore structure of a highly divergent TOM complex of hydrogenosomes in Trichomonas vaginalis

    doi: 10.1371/journal.pbio.3000098

    Figure Lengend Snippet: Localisation and topology of the TA proteins. (A) Immunoblot analysis of TA proteins in T . vaginalis subcellular fractions using α-V5 and α-αSCS (hydrogenosomal matrix protein) antibodies. Total cell lysates, cytoplasm, hydrogenosomes, hydrogenosomes treated with either proteinase K, or hydrogenosomes treated with proteinase K and Triton X-100 isolated from the strains expressing V5-tagged Tom36, Tom46, Homp38, Homp19, and Tom22-like protein. (B) Double transfectants expressing HA-tagged TvTom40-2 along with one of the V5-tagged proteins, Tom36, Tom46, Homp38, Homp19 or Tom22-like protein were visualised using mouse α-HA/α-mouse Abberior STAR 580 (green) and rabbit α-V5/α-rabbit Abberior STAR 635p (red) antibodies. Scale bar, 1 μm. αSCS, α-subunit of succinyl CoA synthetase; CoA, coenzyme A; Cyt, cytoplasm; H/PK, hydrogenosomes treated with proteinase K; H/TX/PK, hydrogenosomes treated with proteinase K in the presence of Triton X-100; HA, human influenza hemagglutinin; Homp, hydrogenosomal outer membrane protein; Hyd, hydrogenosomes; Lys, lysate; TA, tail-anchored; TOM, translocase of the outer membrane; TvTom, T . vaginalis TOM.

    Article Snippet: Isolated hydrogenosomes (protein concentration 1 mg/mL) carrying either HA-tagged or V5-tagged proteins were washed to remove protease inhibitors and incubated for 30 minutes at 37 °C in isolation buffer (225 mM sucrose, 10 mM KH2 PO4 , 20 mM HEPES, 0.5 mM KCl, 5 mM MgCl2 , and 1 mM EDTA [pH 7.2]) supplemented with either 100 μg/mL proteinase K enzyme (Roche Holding AG, Basel, Switzerland) or proteinase K with 0.5% Triton X-100.

    Techniques: Isolation, Expressing, Hemagglutination Assay

    TvTom40-2 was assembled in the mitochondrial outer membrane in  S .  cerevisiae , and it could partially rescue the growth phenotype of  TOM40  mutants. (A) Whole cell lysate and fractions corresponding to cytoplasm and mitochondria were obtained from the WT strain transformed with an empty plasmid (Ø) or a plasmid encoding HA-tagged TvTom40-2. Proteins were analysed by SDS-PAGE and immunodecorated with antibodies against HA, aconitase (mitochondrial matrix protein) and hexokinase (cytosolic protein). (B) The mitochondrial fraction of cells expressing HA-tagged TvTom40-2 were subjected to alkaline extraction. Samples corresponding to supernatant and pellet fractions were analysed by western blotting using antibodies against HA, Aco, and Fis1. (C) Mitochondria as in panel B were treated with proteinase K or with proteinase K in the presence of Triton X-100. Further analysis was as in panel A. (D) Mitochondria were isolated from the strains described in panel A and solubilised in digitonin-containing buffer. Samples were analysed by BN-PAGE and immunodecorated with the indicated antibodies. (E) WT and tet- TOM40  cells transformed with empty plasmid (Ø) or with plasmid encoding either TvTom40-2 or ScTom40 were grown to an OD 600  of 1.0 and spotted in a 1:5 dilution series on synthetic glucose-containing medium lacking Leucine, SD-Leu supplemented with Dox, rich glycerol-containing medium (YPG), or YPG supplemented with Dox. The plates were then incubated at 30 °C for 2 to 3 days. (F) WT strain transformed with empty plasmid (Ø), or  tom40 -25 and  tom40 -34 strains transformed with empty plasmid (Ø) or with a plasmid encoding either TvTom40-2 or ScTom40, were grown to an OD 600  of 1.0 and spotted in a 1:5 dilution series on SD-Leu or YPG. The plates were then incubated at 24 °C, 30 °C, or 37 °C for 2 to 4 days. Aco, aconitase; BN-PAGE, blue native PAGE; Cyt, cytoplasm; Dox, doxycycline; Fis1, mitochondrial fission 1; HA, human influenza hemagglutinin; HK, hexokinase; Lys, lysate; M/PK, mitochondria treated with proteinase K; M/TX/PK, mitochondria treated with proteinase K in the presence of Triton X-100; Mit, mitochondria; OD, optical density; Pel, pellet; SD-Leu, synthetic drop-out medium without leucine; SDS-PAGE, sodium dodecyl sulphate-PAGE; Sup, supernatant; TOM, translocase of the outer membrane; TvTom,  T .  vaginalis  TOM; WT, wild-type; YPG, yeast extract-peptone-glycerol.

    Journal: PLoS Biology

    Article Title: Triplet-pore structure of a highly divergent TOM complex of hydrogenosomes in Trichomonas vaginalis

    doi: 10.1371/journal.pbio.3000098

    Figure Lengend Snippet: TvTom40-2 was assembled in the mitochondrial outer membrane in S . cerevisiae , and it could partially rescue the growth phenotype of TOM40 mutants. (A) Whole cell lysate and fractions corresponding to cytoplasm and mitochondria were obtained from the WT strain transformed with an empty plasmid (Ø) or a plasmid encoding HA-tagged TvTom40-2. Proteins were analysed by SDS-PAGE and immunodecorated with antibodies against HA, aconitase (mitochondrial matrix protein) and hexokinase (cytosolic protein). (B) The mitochondrial fraction of cells expressing HA-tagged TvTom40-2 were subjected to alkaline extraction. Samples corresponding to supernatant and pellet fractions were analysed by western blotting using antibodies against HA, Aco, and Fis1. (C) Mitochondria as in panel B were treated with proteinase K or with proteinase K in the presence of Triton X-100. Further analysis was as in panel A. (D) Mitochondria were isolated from the strains described in panel A and solubilised in digitonin-containing buffer. Samples were analysed by BN-PAGE and immunodecorated with the indicated antibodies. (E) WT and tet- TOM40 cells transformed with empty plasmid (Ø) or with plasmid encoding either TvTom40-2 or ScTom40 were grown to an OD 600 of 1.0 and spotted in a 1:5 dilution series on synthetic glucose-containing medium lacking Leucine, SD-Leu supplemented with Dox, rich glycerol-containing medium (YPG), or YPG supplemented with Dox. The plates were then incubated at 30 °C for 2 to 3 days. (F) WT strain transformed with empty plasmid (Ø), or tom40 -25 and tom40 -34 strains transformed with empty plasmid (Ø) or with a plasmid encoding either TvTom40-2 or ScTom40, were grown to an OD 600 of 1.0 and spotted in a 1:5 dilution series on SD-Leu or YPG. The plates were then incubated at 24 °C, 30 °C, or 37 °C for 2 to 4 days. Aco, aconitase; BN-PAGE, blue native PAGE; Cyt, cytoplasm; Dox, doxycycline; Fis1, mitochondrial fission 1; HA, human influenza hemagglutinin; HK, hexokinase; Lys, lysate; M/PK, mitochondria treated with proteinase K; M/TX/PK, mitochondria treated with proteinase K in the presence of Triton X-100; Mit, mitochondria; OD, optical density; Pel, pellet; SD-Leu, synthetic drop-out medium without leucine; SDS-PAGE, sodium dodecyl sulphate-PAGE; Sup, supernatant; TOM, translocase of the outer membrane; TvTom, T . vaginalis TOM; WT, wild-type; YPG, yeast extract-peptone-glycerol.

    Article Snippet: Isolated hydrogenosomes (protein concentration 1 mg/mL) carrying either HA-tagged or V5-tagged proteins were washed to remove protease inhibitors and incubated for 30 minutes at 37 °C in isolation buffer (225 mM sucrose, 10 mM KH2 PO4 , 20 mM HEPES, 0.5 mM KCl, 5 mM MgCl2 , and 1 mM EDTA [pH 7.2]) supplemented with either 100 μg/mL proteinase K enzyme (Roche Holding AG, Basel, Switzerland) or proteinase K with 0.5% Triton X-100.

    Techniques: Transformation Assay, Plasmid Preparation, Hemagglutination Assay, SDS Page, Expressing, Western Blot, Isolation, Polyacrylamide Gel Electrophoresis, Incubation, Blue Native PAGE

    Localisation of TvTom40-2 in the hydrogenosomal outer membrane. (A) HA-tagged TvTom40-2 and malic enzyme (hydrogenosomal matrix protein) were visualised using mouse α-HA (green) and rabbit α-malic enzyme (red) antibodies, respectively. The nucleus was stained with DAPI (blue). (B) Localisation and topology of TvTom40-2 in  T .  vaginalis  subcellular fractions. Immunoblot analysis of the whole cell lysate, cytoplasm, hydrogenosomes, hydrogenosomes treated with proteinase K, and hydrogenosomes treated with proteinase K in the presence of Triton X-100 using antibodies against HA, Fdx1 (hydrogenosomal matrix protein), and cytosolic malic enzyme. (C) BN-PAGE immunoblots of digitonin-lysed hydrogenosomal extract from the strain expressing HA-tagged TvTom40-2. The samples were probed with α-HA antibody. BN-PAGE, blue native PAGE; Cyt, cytoplasm; cytME, cytoplasmic malic enzyme; DIC, differential interference contrast; Fdx, ferredoxin; H/PK, hydrogenosomes treated with proteinase K; H/TX/PK, hydrogenosomes treated with proteinase K in the presence of Triton X-100; HA, human influenza hemagglutinin; Hyd, hydrogenosomes; Lys, lysate; TOM, translocase of the outer membrane; TvTom,  T .  vaginalis  TOM.

    Journal: PLoS Biology

    Article Title: Triplet-pore structure of a highly divergent TOM complex of hydrogenosomes in Trichomonas vaginalis

    doi: 10.1371/journal.pbio.3000098

    Figure Lengend Snippet: Localisation of TvTom40-2 in the hydrogenosomal outer membrane. (A) HA-tagged TvTom40-2 and malic enzyme (hydrogenosomal matrix protein) were visualised using mouse α-HA (green) and rabbit α-malic enzyme (red) antibodies, respectively. The nucleus was stained with DAPI (blue). (B) Localisation and topology of TvTom40-2 in T . vaginalis subcellular fractions. Immunoblot analysis of the whole cell lysate, cytoplasm, hydrogenosomes, hydrogenosomes treated with proteinase K, and hydrogenosomes treated with proteinase K in the presence of Triton X-100 using antibodies against HA, Fdx1 (hydrogenosomal matrix protein), and cytosolic malic enzyme. (C) BN-PAGE immunoblots of digitonin-lysed hydrogenosomal extract from the strain expressing HA-tagged TvTom40-2. The samples were probed with α-HA antibody. BN-PAGE, blue native PAGE; Cyt, cytoplasm; cytME, cytoplasmic malic enzyme; DIC, differential interference contrast; Fdx, ferredoxin; H/PK, hydrogenosomes treated with proteinase K; H/TX/PK, hydrogenosomes treated with proteinase K in the presence of Triton X-100; HA, human influenza hemagglutinin; Hyd, hydrogenosomes; Lys, lysate; TOM, translocase of the outer membrane; TvTom, T . vaginalis TOM.

    Article Snippet: Isolated hydrogenosomes (protein concentration 1 mg/mL) carrying either HA-tagged or V5-tagged proteins were washed to remove protease inhibitors and incubated for 30 minutes at 37 °C in isolation buffer (225 mM sucrose, 10 mM KH2 PO4 , 20 mM HEPES, 0.5 mM KCl, 5 mM MgCl2 , and 1 mM EDTA [pH 7.2]) supplemented with either 100 μg/mL proteinase K enzyme (Roche Holding AG, Basel, Switzerland) or proteinase K with 0.5% Triton X-100.

    Techniques: Hemagglutination Assay, Staining, Polyacrylamide Gel Electrophoresis, Western Blot, Expressing, Blue Native PAGE