proteinase k Roche Search Results


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  • 99
    Thermo Fisher proteinase k
    Proteinase K, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 21735 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore proteinase k
    Proteinase K, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 26156 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Roche proteinase k roche
    Expression of NICD in cortex and hippocampus and chromatin shearing of the two brain tissues. (A) Sagittal brain sections, view from the midline, display the hippocampus and cortex. Example of immunofluorescence for NICD (red) in (B) cortex and (C) hippocampus of the transgenic mouse line (TNR, for transgenic Notch reporter) expressing enhanced green fluorescent protein (EGFP) in cells with Notch canonical signaling activation. Nuclei are stained in blue. (D) CA hippocampal fields and cortical tissue 1% formaldehyde fixed are sonicated for 30 cycles (30″ ON/30″ OFF) with the Bioruptor ® PLUS at HIGH power setting. All samples were treated with RNase and <t>Proteinase</t> K prior to gel electrophoresis. Scale bar in B and C is 25 μm.
    Proteinase K Roche, supplied by Roche, used in various techniques. Bioz Stars score: 88/100, based on 130 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Anatrace proteinase k
    Deletion of the FHIPEP motif. (A) Motility assay of SJW1103 transformed with pMM130 (encoding wild-type FlhA) and pJM219 (FlhAΔFHIPEP) with and without the addition of 0.1 M IPTG. Transformants were incubated at 30°C for 5 h on semisolid tryptone agar. (B) Extraction of FlhA and FlhAΔFHIPEP in NaOH and Sarkosyl. P, pellet fraction; S, supernatant fraction. (C) Protease protection assay of FlhAΔFHIPEP and HisFLAG-FlhAc. Addition of <t>proteinase</t> K (Prot. K) and CHAPS is indicated by +. Bands of interest are indicated by arrowheads, and positions of molecular weight standards are shown on the left in thousands.
    Proteinase K, supplied by Anatrace, used in various techniques. Bioz Stars score: 92/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Boehringer Mannheim proteinase k
    Deletion of the FHIPEP motif. (A) Motility assay of SJW1103 transformed with pMM130 (encoding wild-type FlhA) and pJM219 (FlhAΔFHIPEP) with and without the addition of 0.1 M IPTG. Transformants were incubated at 30°C for 5 h on semisolid tryptone agar. (B) Extraction of FlhA and FlhAΔFHIPEP in NaOH and Sarkosyl. P, pellet fraction; S, supernatant fraction. (C) Protease protection assay of FlhAΔFHIPEP and HisFLAG-FlhAc. Addition of <t>proteinase</t> K (Prot. K) and CHAPS is indicated by +. Bands of interest are indicated by arrowheads, and positions of molecular weight standards are shown on the left in thousands.
    Proteinase K, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 93/100, based on 2692 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Roche proteinase k phosphostop
    Deletion of the FHIPEP motif. (A) Motility assay of SJW1103 transformed with pMM130 (encoding wild-type FlhA) and pJM219 (FlhAΔFHIPEP) with and without the addition of 0.1 M IPTG. Transformants were incubated at 30°C for 5 h on semisolid tryptone agar. (B) Extraction of FlhA and FlhAΔFHIPEP in NaOH and Sarkosyl. P, pellet fraction; S, supernatant fraction. (C) Protease protection assay of FlhAΔFHIPEP and HisFLAG-FlhAc. Addition of <t>proteinase</t> K (Prot. K) and CHAPS is indicated by +. Bands of interest are indicated by arrowheads, and positions of molecular weight standards are shown on the left in thousands.
    Proteinase K Phosphostop, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Roche recombinant pcr grade proteinase k roche
    Deletion of the FHIPEP motif. (A) Motility assay of SJW1103 transformed with pMM130 (encoding wild-type FlhA) and pJM219 (FlhAΔFHIPEP) with and without the addition of 0.1 M IPTG. Transformants were incubated at 30°C for 5 h on semisolid tryptone agar. (B) Extraction of FlhA and FlhAΔFHIPEP in NaOH and Sarkosyl. P, pellet fraction; S, supernatant fraction. (C) Protease protection assay of FlhAΔFHIPEP and HisFLAG-FlhAc. Addition of <t>proteinase</t> K (Prot. K) and CHAPS is indicated by +. Bands of interest are indicated by arrowheads, and positions of molecular weight standards are shown on the left in thousands.
    Recombinant Pcr Grade Proteinase K Roche, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Roche proteinase k kit
    Deletion of the FHIPEP motif. (A) Motility assay of SJW1103 transformed with pMM130 (encoding wild-type FlhA) and pJM219 (FlhAΔFHIPEP) with and without the addition of 0.1 M IPTG. Transformants were incubated at 30°C for 5 h on semisolid tryptone agar. (B) Extraction of FlhA and FlhAΔFHIPEP in NaOH and Sarkosyl. P, pellet fraction; S, supernatant fraction. (C) Protease protection assay of FlhAΔFHIPEP and HisFLAG-FlhAc. Addition of <t>proteinase</t> K (Prot. K) and CHAPS is indicated by +. Bands of interest are indicated by arrowheads, and positions of molecular weight standards are shown on the left in thousands.
    Proteinase K Kit, supplied by Roche, used in various techniques. Bioz Stars score: 88/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Roche proteinase k solution
    Transport of PfRab5b Q94L -YFP-DD to the cytoplasmic face of infected red blood cells on the TVN. a The selective permeabilization scheme. Saponin permeabilizes RBC plasma membrane (RPM) and PVM, which allows the detection of proteins localized to the outside of the parasite plasma membrane (PPM) or both sides of the PVM. Triton X-100 permeabilizes RPM, PVM, and PPM, allowing staining of the iRBCs, PV, and parasite cytosol with antibody. b Cells expressing PfRab5b Q94L -YFP-DD ( green ) were subjected to an antibody accessibility assay with anti-GFP antibody ( red ) prior to permeabilization with saponin ( upper panel , SAP) or Triton X-100 ( lower panel , Triton). After the saponin treatment, anti-GFP antibody labelled PfRab5b Q94L -YFP-DD secreted to the TVN ( arrowhead ), whereas PfRab5b Q94L -YFP-DD in the parasite cytosol was not labelled. Permeabilization with Triton X-100 allowed labelling of both the parasite cytosolic and TVN-localized PfRab5b Q94L -YFP-DD with anti-GFP antibody. c Schematic of the Shld1 washout assay and the protease accessibility assay. After removal of Shld1, cytosolic DD-tagged proteins are degraded ( red cross and blue circle ) by the parasite proteasome ( yellow cylinder ). DD-tagged proteins, inside of intracellular organelles or transported to the outside of the parasite (blue circular), are resistant to the proteasomal degradation. Since streptolysin O (SLO) permeabilizes RPM but not PVM, DD-tagged proteins in the RBC cytoplasm were selectively degraded by extracellular <t>proteinase</t> K (ProK) after permeabilization with SLO ( black cross and blue circle ). d Sub-cellular localization of PfRab5b Q94L -YFP-DD ( green ) after Shld1 stabilization for 24 h ( upper panel , Shld1) and 2 h after Shld1 washout ( lower panel , washout). After the removal of Shld1, the punctate signal of PfRab5b Q94L -YFP-DD localized to the TR-ceramide ( red )-labelled parasite periphery. Bars 5 µm. e Parasites expressing PfRab5b Q94L -YFP-DD were subjected to Shld1 washout after stabilization (Fig. 3d), and then permeabilized with SLO before treatment with proteinase K. Anti-PfEXP2 and anti-TPx-1 antibodies were used as a control which is not processed with proteinase K, and a loading control for immunoblot analysis, respectively. Representative image from three independent experiments is shown. f The intensity of each band in Fig. 3e were quantitated and the intensity of the band with ProK (+) was divided by that of ProK(−) band. Significance was evaluated by the Student’s t test. Bars standard deviation (n = 3)
    Proteinase K Solution, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 432 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Roche recombinant proteinase k
    Time-dependent peptide degradation in the presence of proteinase K. Percentage of recovery of the input peptides (5 μM) represented as mean ± SD of two replicates, determined by the resulting chromatographic peak area of the corresponding peptide after treatment with <t>proteinase</t> K (5 μg/mL) for different times (0, 1, 2, and 24 h).
    Recombinant Proteinase K, supplied by Roche, used in various techniques. Bioz Stars score: 88/100, based on 102 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Roche pcr grade proteinase k
    Time-dependent peptide degradation in the presence of proteinase K. Percentage of recovery of the input peptides (5 μM) represented as mean ± SD of two replicates, determined by the resulting chromatographic peak area of the corresponding peptide after treatment with <t>proteinase</t> K (5 μg/mL) for different times (0, 1, 2, and 24 h).
    Pcr Grade Proteinase K, supplied by Roche, used in various techniques. Bioz Stars score: 89/100, based on 130 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Roche dna free proteinase k
    Time-dependent peptide degradation in the presence of proteinase K. Percentage of recovery of the input peptides (5 μM) represented as mean ± SD of two replicates, determined by the resulting chromatographic peak area of the corresponding peptide after treatment with <t>proteinase</t> K (5 μg/mL) for different times (0, 1, 2, and 24 h).
    Dna Free Proteinase K, supplied by Roche, used in various techniques. Bioz Stars score: 88/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche rnase free proteinase k
    Time-dependent peptide degradation in the presence of proteinase K. Percentage of recovery of the input peptides (5 μM) represented as mean ± SD of two replicates, determined by the resulting chromatographic peak area of the corresponding peptide after treatment with <t>proteinase</t> K (5 μg/mL) for different times (0, 1, 2, and 24 h).
    Rnase Free Proteinase K, supplied by Roche, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Roche proteinase k dna isolation
    Time-dependent peptide degradation in the presence of proteinase K. Percentage of recovery of the input peptides (5 μM) represented as mean ± SD of two replicates, determined by the resulting chromatographic peak area of the corresponding peptide after treatment with <t>proteinase</t> K (5 μg/mL) for different times (0, 1, 2, and 24 h).
    Proteinase K Dna Isolation, supplied by Roche, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Roche proteinase k nuclease free buffer
    Time-dependent peptide degradation in the presence of proteinase K. Percentage of recovery of the input peptides (5 μM) represented as mean ± SD of two replicates, determined by the resulting chromatographic peak area of the corresponding peptide after treatment with <t>proteinase</t> K (5 μg/mL) for different times (0, 1, 2, and 24 h).
    Proteinase K Nuclease Free Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche nahco3 proteinase k
    Time-dependent peptide degradation in the presence of proteinase K. Percentage of recovery of the input peptides (5 μM) represented as mean ± SD of two replicates, determined by the resulting chromatographic peak area of the corresponding peptide after treatment with <t>proteinase</t> K (5 μg/mL) for different times (0, 1, 2, and 24 h).
    Nahco3 Proteinase K, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Roche 20ul proteinase k
    Time-dependent peptide degradation in the presence of proteinase K. Percentage of recovery of the input peptides (5 μM) represented as mean ± SD of two replicates, determined by the resulting chromatographic peak area of the corresponding peptide after treatment with <t>proteinase</t> K (5 μg/mL) for different times (0, 1, 2, and 24 h).
    20ul Proteinase K, supplied by Roche, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Expression of NICD in cortex and hippocampus and chromatin shearing of the two brain tissues. (A) Sagittal brain sections, view from the midline, display the hippocampus and cortex. Example of immunofluorescence for NICD (red) in (B) cortex and (C) hippocampus of the transgenic mouse line (TNR, for transgenic Notch reporter) expressing enhanced green fluorescent protein (EGFP) in cells with Notch canonical signaling activation. Nuclei are stained in blue. (D) CA hippocampal fields and cortical tissue 1% formaldehyde fixed are sonicated for 30 cycles (30″ ON/30″ OFF) with the Bioruptor ® PLUS at HIGH power setting. All samples were treated with RNase and Proteinase K prior to gel electrophoresis. Scale bar in B and C is 25 μm.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: TF-ChIP Method for Tissue-Specific Gene Targets

    doi: 10.3389/fncel.2019.00095

    Figure Lengend Snippet: Expression of NICD in cortex and hippocampus and chromatin shearing of the two brain tissues. (A) Sagittal brain sections, view from the midline, display the hippocampus and cortex. Example of immunofluorescence for NICD (red) in (B) cortex and (C) hippocampus of the transgenic mouse line (TNR, for transgenic Notch reporter) expressing enhanced green fluorescent protein (EGFP) in cells with Notch canonical signaling activation. Nuclei are stained in blue. (D) CA hippocampal fields and cortical tissue 1% formaldehyde fixed are sonicated for 30 cycles (30″ ON/30″ OFF) with the Bioruptor ® PLUS at HIGH power setting. All samples were treated with RNase and Proteinase K prior to gel electrophoresis. Scale bar in B and C is 25 μm.

    Article Snippet: Ethylenediaminetetraacetic acid (EDTA) (Sigma–Aldrich, United States; E5134) Tris (Roth, Germany; 5429.3) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Sigma–Aldrich, United States; H3375) NaCl (Roth, Germany; 9265.2) Sodium deoxycholate (Sigma–Aldrich, United States; 30970) Triton X-100 (Sigma–Aldrich, United States; 93426) Tris–HCl (Roth, Germany; 9090) LiCl (Sigma–Aldrich, United States; L9650) Nonidet P-40 substitute (Sigma–Aldrich, United States; 74385) QubitTM dsDNA HS Assay Kit (Thermo Fisher, United States; Q32851) PierceTM Protein A/G Agarose (Thermo Fisher, United States; 20421) Antibodies: Rabbit anti-NICD (Cell Signaling, United States; #4147); Rabbit IgG (Cell Signaling, United States; #2729); and Rabbit anti-Acetyl-Histone H3 (Lys9) (Cell Signaling, United States; #9649) RNase A solution (Promega, United States; A7974) Proteinase K (Roche, Switzerland; 03508838103) Phenol/chloroform/isoamyl alcohol (Roth, Germany; A156) !Caution chloroform is toxic if absorbed through the skin, inhaled or ingested.

    Techniques: Expressing, Immunofluorescence, Transgenic Assay, Activation Assay, Staining, Sonication, Nucleic Acid Electrophoresis

    PUSL1 Is a Mitochondrial Matrix Protein and Interacts with the Mitoribosome (A) Western blot analysis of PUSL1-FLAG expression induced by tetracycline (at 0, 10, 50, or 100 ng/mL final concentration) in control and PUSL1-FLAG HEK293T cells. SDHA is used as a loading control. (B) Protease protection assay to assess submitochondrial localization of proteins. Mitochondria (Mito., non-treated) were swollen in hypotonic buffer (Swell.) or lysed with 1% triton X-100-supplemented buffer (triton). Samples were left untreated (−) or treated (+) with 50 μg/mL proteinase K (PK). (C) Western blot analysis of mitochondrial proteins incubated in HEPES buffer (negative control) or sodium carbonate at indicated pH values. Total (T), pellet (P), and supernatant (S) correspond to fractions obtained before and after extraction and centrifugation. (D) LFQ-MS/MS of co-immunoprecipitated PUSL1-FLAG-associated complexes using digitonin to lyse the mitochondria of HEK293T cells expressing PUSL1-FLAG (n = 4) or Mito-GFP (control, n = 3). Mitoribosomal proteins are highlighted in green (SSU) and blue (LSU). Translation-associated proteins are highlighted in orange and other significantly enriched proteins are highlighted in red. The x axis represents the fold change and the y axis indicates the adjusted p value of PUSL1-FLAG versus Mito-GFP. The dashed line represents a 5% false discovery rate. See also Tables S1 and S2 .

    Journal: Cell Reports

    Article Title: MitoRibo-Tag Mice Provide a Tool for In Vivo Studies of Mitoribosome Composition

    doi: 10.1016/j.celrep.2019.09.080

    Figure Lengend Snippet: PUSL1 Is a Mitochondrial Matrix Protein and Interacts with the Mitoribosome (A) Western blot analysis of PUSL1-FLAG expression induced by tetracycline (at 0, 10, 50, or 100 ng/mL final concentration) in control and PUSL1-FLAG HEK293T cells. SDHA is used as a loading control. (B) Protease protection assay to assess submitochondrial localization of proteins. Mitochondria (Mito., non-treated) were swollen in hypotonic buffer (Swell.) or lysed with 1% triton X-100-supplemented buffer (triton). Samples were left untreated (−) or treated (+) with 50 μg/mL proteinase K (PK). (C) Western blot analysis of mitochondrial proteins incubated in HEPES buffer (negative control) or sodium carbonate at indicated pH values. Total (T), pellet (P), and supernatant (S) correspond to fractions obtained before and after extraction and centrifugation. (D) LFQ-MS/MS of co-immunoprecipitated PUSL1-FLAG-associated complexes using digitonin to lyse the mitochondria of HEK293T cells expressing PUSL1-FLAG (n = 4) or Mito-GFP (control, n = 3). Mitoribosomal proteins are highlighted in green (SSU) and blue (LSU). Translation-associated proteins are highlighted in orange and other significantly enriched proteins are highlighted in red. The x axis represents the fold change and the y axis indicates the adjusted p value of PUSL1-FLAG versus Mito-GFP. The dashed line represents a 5% false discovery rate. See also Tables S1 and S2 .

    Article Snippet: The aliquots were split into two, whereby to one aliquot proteinase K (Roche, cat. no. 03450384103) was added to a final concentration of 25 μg/ml.

    Techniques: Western Blot, Expressing, Concentration Assay, Incubation, Negative Control, Centrifugation, Mass Spectrometry, Immunoprecipitation

    Adaptation of the infectious recombinant prion (L-seeded-PMCA) to the new in vitro propagation method (L-seeded-PMSA). A) Immunodetection of the characteristic protease resistant core resulting from cleavage of the amino-terminal tail of the original PMCA-derived recombinant prions (H/L-seeded-03 and L-seeded-PMCA) and the PMSA-adapted one (L-seeded-PMSA) by Western blotting. Samples were digested with 85 μg/ml of proteinase-K (PK) and analyzed by Western blotting using monoclonal antibodies 12B2 (1:2,500) and D18 (1:5,000). Differential digestion of the N-terminal makes the two recombinant prions adapted to propagate in dextran sulfate-complemented substrates (L-seeded-PMCA and L-seeded-PMSA) undetectable by 12B2 mAb (epitope 88–92) whereas the Prnp 0/0 -complemented sample (H/L-seeded-03), which according to previous results (10) is a mixture of strains, detectable by both 12B2 and D18 mAbs (epitope 143–149). B) Electrophoretic migration patterns of L-seeded-PMCA and L-seeded-PMSA recombinant prions. The two recombinant prions were digested with PK and analyzed by BlueSafe staining (total protein) to examine all the proteolytic fragments derived from digestion. The same gel was transferred for Western blotting and developed with D18 mAb. Both recombinant misfolded PrPs show a similar banding pattern, suggesting similar if not identical conformations. The differences on electrophoretic patterns detected by D18 antibody between the gel in panel A and the one in panel B are due to the amount of sample loaded in each case. For total protein staining and posterior western blotting from panel B, 50x more sample is loaded than for panel A, making some minor PrP fragments detectable. C) Determination of potential infectivity (ability to propagate on brain-derived PrP) of L-seeded-PMCA and L-seeded-PMSA recombinant prions by brain-PMCA. Conservation of the capacity to misfold brain-derived PrP C , as an indication of potential in vivo infectivity, was evaluated using equal amounts of the two recombinant inocula as seeds in TgVole brain homogenate at 1:10 dilution during one round of PMCA. Triplicates (T1, T2 T3) were performed for both samples and PK-resistant PrP formation was monitored by Western blotting with D18 mAb. Misfolded brain-derived PrP arises from the first round for the dextran sulfate-complemented seeds with no difference between PMCA- or PMSA-adapted seeds (L-seeded-PMCA and L-seeded-PMSA). (→) Signals of PK-digested misfolded rec-PrPs appear lower than those of the non-glycosylated band due to the absence of the GPI anchor. rec-PrP (control): undigested bank vole rec-PrP protein. Brain (control): undigested TgVole whole brain homogenate. NA: Non-amplified samples.

    Journal: PLoS Pathogens

    Article Title: Development of a new largely scalable in vitro prion propagation method for the production of infectious recombinant prions for high resolution structural studies

    doi: 10.1371/journal.ppat.1008117

    Figure Lengend Snippet: Adaptation of the infectious recombinant prion (L-seeded-PMCA) to the new in vitro propagation method (L-seeded-PMSA). A) Immunodetection of the characteristic protease resistant core resulting from cleavage of the amino-terminal tail of the original PMCA-derived recombinant prions (H/L-seeded-03 and L-seeded-PMCA) and the PMSA-adapted one (L-seeded-PMSA) by Western blotting. Samples were digested with 85 μg/ml of proteinase-K (PK) and analyzed by Western blotting using monoclonal antibodies 12B2 (1:2,500) and D18 (1:5,000). Differential digestion of the N-terminal makes the two recombinant prions adapted to propagate in dextran sulfate-complemented substrates (L-seeded-PMCA and L-seeded-PMSA) undetectable by 12B2 mAb (epitope 88–92) whereas the Prnp 0/0 -complemented sample (H/L-seeded-03), which according to previous results (10) is a mixture of strains, detectable by both 12B2 and D18 mAbs (epitope 143–149). B) Electrophoretic migration patterns of L-seeded-PMCA and L-seeded-PMSA recombinant prions. The two recombinant prions were digested with PK and analyzed by BlueSafe staining (total protein) to examine all the proteolytic fragments derived from digestion. The same gel was transferred for Western blotting and developed with D18 mAb. Both recombinant misfolded PrPs show a similar banding pattern, suggesting similar if not identical conformations. The differences on electrophoretic patterns detected by D18 antibody between the gel in panel A and the one in panel B are due to the amount of sample loaded in each case. For total protein staining and posterior western blotting from panel B, 50x more sample is loaded than for panel A, making some minor PrP fragments detectable. C) Determination of potential infectivity (ability to propagate on brain-derived PrP) of L-seeded-PMCA and L-seeded-PMSA recombinant prions by brain-PMCA. Conservation of the capacity to misfold brain-derived PrP C , as an indication of potential in vivo infectivity, was evaluated using equal amounts of the two recombinant inocula as seeds in TgVole brain homogenate at 1:10 dilution during one round of PMCA. Triplicates (T1, T2 T3) were performed for both samples and PK-resistant PrP formation was monitored by Western blotting with D18 mAb. Misfolded brain-derived PrP arises from the first round for the dextran sulfate-complemented seeds with no difference between PMCA- or PMSA-adapted seeds (L-seeded-PMCA and L-seeded-PMSA). (→) Signals of PK-digested misfolded rec-PrPs appear lower than those of the non-glycosylated band due to the absence of the GPI anchor. rec-PrP (control): undigested bank vole rec-PrP protein. Brain (control): undigested TgVole whole brain homogenate. NA: Non-amplified samples.

    Article Snippet: Biochemical characterization of in vitro - and in vivo -generated prion strains Protease K digestion : recPMCA products based on Prnp 0/0 complemented substrates were digested by mixing the sample with N-Lauroylsarcosine sodium salt (sarkosyl) (Sigma-Aldrich) 10% in PBS 1:1 (v/v) and adding proteinase K (PK) (Roche) at 85 μg/ml for 1 h at 42°C and shaking at 450 rpm as described previously [ ].

    Techniques: Recombinant, In Vitro, Immunodetection, Derivative Assay, Western Blot, Migration, Staining, Infection, In Vivo, Amplification

    Biochemical characterization of L-seeded-PMSA. A) The ability of L-seeded-PMSA to self-propagate indefinitely shown through 30 serial rounds of PMSA at 1:1,000 dilution. 500 μl of the original seed (10 0 dilution), the product of round 17 of PMSA (10 −50 dilution) and the product of round 33 (10 −100 dilution) were PK-digested, concentrated by centrifugation and visualized by total protein staining and showed no detectable pattern differences throughout the propagation process. B) Protease-K (PK) resistance of L-seeded PMSA was evaluated by submitting fractions of the same sample to increasing PK concentrations for 1h at 42°C. L-seeded-PMSA showed high resistance to protease, as the main PK-resistant bands are maintained even at the highest PK concentration used. C) Identification of the proteolytic fragments of L-seeded-PMSA by epitope mapping was performed with the same PK-digested and concentrated sample divided in seven that were stained for total protein to guarantee equal protein amounts in each lane. The stained gel was then transferred for Western blotting and the following antibodies were used for epitope mapping: 12B2 (1:2,500), 9A2 (1:4,000), 7D9 (1:1,000), D18 (1:5,000), Saf83 (1:400), Sa84 (1:400) and POM19 (1:10,000). The theoretical reconstruction of the PrP fragments recognized by each antibody are represented in the cartoon, the most abundant fragments were (see arrows) ~15 (pink), ~9 (purple) and ~2 (orange) kDa. Another two minor bands reflect other PK cleavages yielding two fragments of ~12 and ~6 kDa (green and red bands, respectively). These fragments are not always detected by all the antibodies used for the epitope mapping due to their low amounts, below the detection limit of some of the antibodies. MW: Molecular weight.

    Journal: PLoS Pathogens

    Article Title: Development of a new largely scalable in vitro prion propagation method for the production of infectious recombinant prions for high resolution structural studies

    doi: 10.1371/journal.ppat.1008117

    Figure Lengend Snippet: Biochemical characterization of L-seeded-PMSA. A) The ability of L-seeded-PMSA to self-propagate indefinitely shown through 30 serial rounds of PMSA at 1:1,000 dilution. 500 μl of the original seed (10 0 dilution), the product of round 17 of PMSA (10 −50 dilution) and the product of round 33 (10 −100 dilution) were PK-digested, concentrated by centrifugation and visualized by total protein staining and showed no detectable pattern differences throughout the propagation process. B) Protease-K (PK) resistance of L-seeded PMSA was evaluated by submitting fractions of the same sample to increasing PK concentrations for 1h at 42°C. L-seeded-PMSA showed high resistance to protease, as the main PK-resistant bands are maintained even at the highest PK concentration used. C) Identification of the proteolytic fragments of L-seeded-PMSA by epitope mapping was performed with the same PK-digested and concentrated sample divided in seven that were stained for total protein to guarantee equal protein amounts in each lane. The stained gel was then transferred for Western blotting and the following antibodies were used for epitope mapping: 12B2 (1:2,500), 9A2 (1:4,000), 7D9 (1:1,000), D18 (1:5,000), Saf83 (1:400), Sa84 (1:400) and POM19 (1:10,000). The theoretical reconstruction of the PrP fragments recognized by each antibody are represented in the cartoon, the most abundant fragments were (see arrows) ~15 (pink), ~9 (purple) and ~2 (orange) kDa. Another two minor bands reflect other PK cleavages yielding two fragments of ~12 and ~6 kDa (green and red bands, respectively). These fragments are not always detected by all the antibodies used for the epitope mapping due to their low amounts, below the detection limit of some of the antibodies. MW: Molecular weight.

    Article Snippet: Biochemical characterization of in vitro - and in vivo -generated prion strains Protease K digestion : recPMCA products based on Prnp 0/0 complemented substrates were digested by mixing the sample with N-Lauroylsarcosine sodium salt (sarkosyl) (Sigma-Aldrich) 10% in PBS 1:1 (v/v) and adding proteinase K (PK) (Roche) at 85 μg/ml for 1 h at 42°C and shaking at 450 rpm as described previously [ ].

    Techniques: Centrifugation, Staining, Concentration Assay, Western Blot, Molecular Weight

    PrP Sc detection and histopathological analysis of diseased bank vole 109I brains inoculated with CWD-vole, L-seeded-PMCA and L-seeded-PMSA. A) Biochemical analysis of Proteinase-K (PK)-resistant PrP Sc in brain homogenates from bank vole I109 inoculated with the misfolded rec-PrPs: L-seeded-PMCA and L-seeded-PMSA, and CWD-vole as control. Representative bank vole brain homogenates were digested with PK (200 μg/ml). All rec-PrP Sc inoculated bank vole brains accumulated a classical PrP Sc type characterized by a three-banded electrophoretic migration pattern, indistinguishable from the CWD-vole inoculated vole brains. 9A2 monoclonal antibody (1:5,000). Control: undigested bank vole whole brain homogenate. MW: Molecular weight. B) Brain deposition of PrP Sc in the mediodorsal thalamic nucleus and the hippocampus of bank voles inoculated with L-seeded-PMCA or L-seeded-PMSA was assessed by immunohistochemistry using the monoclonal antibody 6C2 (1:300). Note, with both seeds, deposition of PrP Sc could be observed in the mediodorsal thalamic nucleus but not in the hippocampus.

    Journal: PLoS Pathogens

    Article Title: Development of a new largely scalable in vitro prion propagation method for the production of infectious recombinant prions for high resolution structural studies

    doi: 10.1371/journal.ppat.1008117

    Figure Lengend Snippet: PrP Sc detection and histopathological analysis of diseased bank vole 109I brains inoculated with CWD-vole, L-seeded-PMCA and L-seeded-PMSA. A) Biochemical analysis of Proteinase-K (PK)-resistant PrP Sc in brain homogenates from bank vole I109 inoculated with the misfolded rec-PrPs: L-seeded-PMCA and L-seeded-PMSA, and CWD-vole as control. Representative bank vole brain homogenates were digested with PK (200 μg/ml). All rec-PrP Sc inoculated bank vole brains accumulated a classical PrP Sc type characterized by a three-banded electrophoretic migration pattern, indistinguishable from the CWD-vole inoculated vole brains. 9A2 monoclonal antibody (1:5,000). Control: undigested bank vole whole brain homogenate. MW: Molecular weight. B) Brain deposition of PrP Sc in the mediodorsal thalamic nucleus and the hippocampus of bank voles inoculated with L-seeded-PMCA or L-seeded-PMSA was assessed by immunohistochemistry using the monoclonal antibody 6C2 (1:300). Note, with both seeds, deposition of PrP Sc could be observed in the mediodorsal thalamic nucleus but not in the hippocampus.

    Article Snippet: Biochemical characterization of in vitro - and in vivo -generated prion strains Protease K digestion : recPMCA products based on Prnp 0/0 complemented substrates were digested by mixing the sample with N-Lauroylsarcosine sodium salt (sarkosyl) (Sigma-Aldrich) 10% in PBS 1:1 (v/v) and adding proteinase K (PK) (Roche) at 85 μg/ml for 1 h at 42°C and shaking at 450 rpm as described previously [ ].

    Techniques: Migration, Molecular Weight, Immunohistochemistry

    PrP Sc detection in diseased TgVole (1x) brains inoculated with CWD-vole, H/L-seeded-03, L-seeded-PMCA, L-seeded-PMSA, non-infectious fibrillary rec-PrP and non-fibrillated rec-PrP. Biochemical analysis of Proteinase-K (PK)-resistant PrP Sc in brain homogenates from TgVole (1x) inoculated with different misfolded PrPs: CWD-vole, H/L-seeded-03, L-seeded-PMCA, L-seeded-PMSA (I II), non-infectious fibrillary rec-PrP and non-fibrillated rec-PrP. Representative TgVole brain homogenates were digested with 200 μg/ml of PK. The CWD-vole and the PMCA and PMSA-derived misfolded rec-PrPs inoculated TgVole brains accumulated a classical PrP Sc type characterized by a three bands electrophoretic migration pattern. Brains from animals inoculated with the non-infectious fibrillary rec-PrP and non-fibrillated rec-PrP, used as negative controls, did not show any PK resistant band. D18 monoclonal antibody (1:5,000). Control: undigested TgVole (1x) whole brain homogenate. MW: Molecular weight. dpi: days post-inoculation at which each animal was culled due to neurological clinical signs.

    Journal: PLoS Pathogens

    Article Title: Development of a new largely scalable in vitro prion propagation method for the production of infectious recombinant prions for high resolution structural studies

    doi: 10.1371/journal.ppat.1008117

    Figure Lengend Snippet: PrP Sc detection in diseased TgVole (1x) brains inoculated with CWD-vole, H/L-seeded-03, L-seeded-PMCA, L-seeded-PMSA, non-infectious fibrillary rec-PrP and non-fibrillated rec-PrP. Biochemical analysis of Proteinase-K (PK)-resistant PrP Sc in brain homogenates from TgVole (1x) inoculated with different misfolded PrPs: CWD-vole, H/L-seeded-03, L-seeded-PMCA, L-seeded-PMSA (I II), non-infectious fibrillary rec-PrP and non-fibrillated rec-PrP. Representative TgVole brain homogenates were digested with 200 μg/ml of PK. The CWD-vole and the PMCA and PMSA-derived misfolded rec-PrPs inoculated TgVole brains accumulated a classical PrP Sc type characterized by a three bands electrophoretic migration pattern. Brains from animals inoculated with the non-infectious fibrillary rec-PrP and non-fibrillated rec-PrP, used as negative controls, did not show any PK resistant band. D18 monoclonal antibody (1:5,000). Control: undigested TgVole (1x) whole brain homogenate. MW: Molecular weight. dpi: days post-inoculation at which each animal was culled due to neurological clinical signs.

    Article Snippet: Biochemical characterization of in vitro - and in vivo -generated prion strains Protease K digestion : recPMCA products based on Prnp 0/0 complemented substrates were digested by mixing the sample with N-Lauroylsarcosine sodium salt (sarkosyl) (Sigma-Aldrich) 10% in PBS 1:1 (v/v) and adding proteinase K (PK) (Roche) at 85 μg/ml for 1 h at 42°C and shaking at 450 rpm as described previously [ ].

    Techniques: Derivative Assay, Migration, Molecular Weight

    Deletion of the FHIPEP motif. (A) Motility assay of SJW1103 transformed with pMM130 (encoding wild-type FlhA) and pJM219 (FlhAΔFHIPEP) with and without the addition of 0.1 M IPTG. Transformants were incubated at 30°C for 5 h on semisolid tryptone agar. (B) Extraction of FlhA and FlhAΔFHIPEP in NaOH and Sarkosyl. P, pellet fraction; S, supernatant fraction. (C) Protease protection assay of FlhAΔFHIPEP and HisFLAG-FlhAc. Addition of proteinase K (Prot. K) and CHAPS is indicated by +. Bands of interest are indicated by arrowheads, and positions of molecular weight standards are shown on the left in thousands.

    Journal: Journal of Bacteriology

    Article Title: Analysis of the Cytoplasmic Domains of Salmonella FlhA and Interactions with Components of the Flagellar Export Machinery

    doi: 10.1128/JB.186.22.7586-7592.2004

    Figure Lengend Snippet: Deletion of the FHIPEP motif. (A) Motility assay of SJW1103 transformed with pMM130 (encoding wild-type FlhA) and pJM219 (FlhAΔFHIPEP) with and without the addition of 0.1 M IPTG. Transformants were incubated at 30°C for 5 h on semisolid tryptone agar. (B) Extraction of FlhA and FlhAΔFHIPEP in NaOH and Sarkosyl. P, pellet fraction; S, supernatant fraction. (C) Protease protection assay of FlhAΔFHIPEP and HisFLAG-FlhAc. Addition of proteinase K (Prot. K) and CHAPS is indicated by +. Bands of interest are indicated by arrowheads, and positions of molecular weight standards are shown on the left in thousands.

    Article Snippet: Three aliquots were taken: one was a no-treatment control, another was treated with 25 μg of proteinase K (Roche, Mannheim, Germany) per ml, and the last was treated with the same concentration of proteinase K in the presence of 0.2% CHAPS (Anatrace, Maumee, Ohio) to solubilize the membranes and allow the protease access to cytoplasmic components.

    Techniques: Motility Assay, Transformation Assay, Incubation, Molecular Weight

    Deletion of 18QWQIL22 and amino-terminal sequence. (A) Motility assay of SJW1364 and SJW1103 transformed with pMM108 (encoding His-FLAG-FlhA), pJM272 (His-FlhAΔQWQIL), or pJM273 (His-FlhAΔNT). Transformants were incubated at 30°C for 5 h on semisolid tryptone agar. (B) Extraction of His-FlhA, His-FlhAΔQWQIL, and His-FlhAΔNT in NaOH and Sarkosyl. P, pellet fraction; S, supernatant fraction. (C) Protease protection assay of His-FlhA, His-FlhAΔQWQIL, and His-FlhAΔNT. Addition of proteinase K (Prot. K) and CHAPS is indicated by +. Bands of interest are indicated by arrowheads, and positions of molecular weight standards are shown on the left in thousands.

    Journal: Journal of Bacteriology

    Article Title: Analysis of the Cytoplasmic Domains of Salmonella FlhA and Interactions with Components of the Flagellar Export Machinery

    doi: 10.1128/JB.186.22.7586-7592.2004

    Figure Lengend Snippet: Deletion of 18QWQIL22 and amino-terminal sequence. (A) Motility assay of SJW1364 and SJW1103 transformed with pMM108 (encoding His-FLAG-FlhA), pJM272 (His-FlhAΔQWQIL), or pJM273 (His-FlhAΔNT). Transformants were incubated at 30°C for 5 h on semisolid tryptone agar. (B) Extraction of His-FlhA, His-FlhAΔQWQIL, and His-FlhAΔNT in NaOH and Sarkosyl. P, pellet fraction; S, supernatant fraction. (C) Protease protection assay of His-FlhA, His-FlhAΔQWQIL, and His-FlhAΔNT. Addition of proteinase K (Prot. K) and CHAPS is indicated by +. Bands of interest are indicated by arrowheads, and positions of molecular weight standards are shown on the left in thousands.

    Article Snippet: Three aliquots were taken: one was a no-treatment control, another was treated with 25 μg of proteinase K (Roche, Mannheim, Germany) per ml, and the last was treated with the same concentration of proteinase K in the presence of 0.2% CHAPS (Anatrace, Maumee, Ohio) to solubilize the membranes and allow the protease access to cytoplasmic components.

    Techniques: Sequencing, Motility Assay, Transformation Assay, Incubation, Molecular Weight

    Transport of PfRab5b Q94L -YFP-DD to the cytoplasmic face of infected red blood cells on the TVN. a The selective permeabilization scheme. Saponin permeabilizes RBC plasma membrane (RPM) and PVM, which allows the detection of proteins localized to the outside of the parasite plasma membrane (PPM) or both sides of the PVM. Triton X-100 permeabilizes RPM, PVM, and PPM, allowing staining of the iRBCs, PV, and parasite cytosol with antibody. b Cells expressing PfRab5b Q94L -YFP-DD ( green ) were subjected to an antibody accessibility assay with anti-GFP antibody ( red ) prior to permeabilization with saponin ( upper panel , SAP) or Triton X-100 ( lower panel , Triton). After the saponin treatment, anti-GFP antibody labelled PfRab5b Q94L -YFP-DD secreted to the TVN ( arrowhead ), whereas PfRab5b Q94L -YFP-DD in the parasite cytosol was not labelled. Permeabilization with Triton X-100 allowed labelling of both the parasite cytosolic and TVN-localized PfRab5b Q94L -YFP-DD with anti-GFP antibody. c Schematic of the Shld1 washout assay and the protease accessibility assay. After removal of Shld1, cytosolic DD-tagged proteins are degraded ( red cross and blue circle ) by the parasite proteasome ( yellow cylinder ). DD-tagged proteins, inside of intracellular organelles or transported to the outside of the parasite (blue circular), are resistant to the proteasomal degradation. Since streptolysin O (SLO) permeabilizes RPM but not PVM, DD-tagged proteins in the RBC cytoplasm were selectively degraded by extracellular proteinase K (ProK) after permeabilization with SLO ( black cross and blue circle ). d Sub-cellular localization of PfRab5b Q94L -YFP-DD ( green ) after Shld1 stabilization for 24 h ( upper panel , Shld1) and 2 h after Shld1 washout ( lower panel , washout). After the removal of Shld1, the punctate signal of PfRab5b Q94L -YFP-DD localized to the TR-ceramide ( red )-labelled parasite periphery. Bars 5 µm. e Parasites expressing PfRab5b Q94L -YFP-DD were subjected to Shld1 washout after stabilization (Fig. 3d), and then permeabilized with SLO before treatment with proteinase K. Anti-PfEXP2 and anti-TPx-1 antibodies were used as a control which is not processed with proteinase K, and a loading control for immunoblot analysis, respectively. Representative image from three independent experiments is shown. f The intensity of each band in Fig. 3e were quantitated and the intensity of the band with ProK (+) was divided by that of ProK(−) band. Significance was evaluated by the Student’s t test. Bars standard deviation (n = 3)

    Journal: Malaria Journal

    Article Title: Plasmodium Rab5b is secreted to the cytoplasmic face of the tubovesicular network in infected red blood cells together with N-acylated adenylate kinase 2

    doi: 10.1186/s12936-016-1377-4

    Figure Lengend Snippet: Transport of PfRab5b Q94L -YFP-DD to the cytoplasmic face of infected red blood cells on the TVN. a The selective permeabilization scheme. Saponin permeabilizes RBC plasma membrane (RPM) and PVM, which allows the detection of proteins localized to the outside of the parasite plasma membrane (PPM) or both sides of the PVM. Triton X-100 permeabilizes RPM, PVM, and PPM, allowing staining of the iRBCs, PV, and parasite cytosol with antibody. b Cells expressing PfRab5b Q94L -YFP-DD ( green ) were subjected to an antibody accessibility assay with anti-GFP antibody ( red ) prior to permeabilization with saponin ( upper panel , SAP) or Triton X-100 ( lower panel , Triton). After the saponin treatment, anti-GFP antibody labelled PfRab5b Q94L -YFP-DD secreted to the TVN ( arrowhead ), whereas PfRab5b Q94L -YFP-DD in the parasite cytosol was not labelled. Permeabilization with Triton X-100 allowed labelling of both the parasite cytosolic and TVN-localized PfRab5b Q94L -YFP-DD with anti-GFP antibody. c Schematic of the Shld1 washout assay and the protease accessibility assay. After removal of Shld1, cytosolic DD-tagged proteins are degraded ( red cross and blue circle ) by the parasite proteasome ( yellow cylinder ). DD-tagged proteins, inside of intracellular organelles or transported to the outside of the parasite (blue circular), are resistant to the proteasomal degradation. Since streptolysin O (SLO) permeabilizes RPM but not PVM, DD-tagged proteins in the RBC cytoplasm were selectively degraded by extracellular proteinase K (ProK) after permeabilization with SLO ( black cross and blue circle ). d Sub-cellular localization of PfRab5b Q94L -YFP-DD ( green ) after Shld1 stabilization for 24 h ( upper panel , Shld1) and 2 h after Shld1 washout ( lower panel , washout). After the removal of Shld1, the punctate signal of PfRab5b Q94L -YFP-DD localized to the TR-ceramide ( red )-labelled parasite periphery. Bars 5 µm. e Parasites expressing PfRab5b Q94L -YFP-DD were subjected to Shld1 washout after stabilization (Fig. 3d), and then permeabilized with SLO before treatment with proteinase K. Anti-PfEXP2 and anti-TPx-1 antibodies were used as a control which is not processed with proteinase K, and a loading control for immunoblot analysis, respectively. Representative image from three independent experiments is shown. f The intensity of each band in Fig. 3e were quantitated and the intensity of the band with ProK (+) was divided by that of ProK(−) band. Significance was evaluated by the Student’s t test. Bars standard deviation (n = 3)

    Article Snippet: Proteinase K solution (1 µl, 18 mg/ml, Roche 11389200) was added to one tube, and both tubes were incubated on ice for 30 min. Phenylmethylsulfonyl fluoride (PMSF) (5 µl, 200 mM) was added to each sample, followed by 25 µl of 3 × SDS-PAGE buffer, and then samples were incubated at 95 °C for 5 min. Fifteen-microliter samples were loaded into each well of the SDS-PAGE gels.

    Techniques: Infection, Staining, Expressing, Standard Deviation

    Time-dependent peptide degradation in the presence of proteinase K. Percentage of recovery of the input peptides (5 μM) represented as mean ± SD of two replicates, determined by the resulting chromatographic peak area of the corresponding peptide after treatment with proteinase K (5 μg/mL) for different times (0, 1, 2, and 24 h).

    Journal: Frontiers in Microbiology

    Article Title: Mapping and Identification of Antifungal Peptides in the Putative Antifungal Protein AfpB from the Filamentous Fungus Penicillium digitatum

    doi: 10.3389/fmicb.2017.00592

    Figure Lengend Snippet: Time-dependent peptide degradation in the presence of proteinase K. Percentage of recovery of the input peptides (5 μM) represented as mean ± SD of two replicates, determined by the resulting chromatographic peak area of the corresponding peptide after treatment with proteinase K (5 μg/mL) for different times (0, 1, 2, and 24 h).

    Article Snippet: Peptides (5 μM) were dissolved in 10 mM MOPS pH 7 and digested with 5 μg/mL of recombinant proteinase K (2 U/mg; Roche, Mannheim, Germany) at 30°C.

    Techniques: