streptavidin horseradish peroxidase  (Thermo Fisher)


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    Structured Review

    Thermo Fisher streptavidin horseradish peroxidase
    Expression of protocadherins. GFP was electroporated alone or in combination with protocadherins into K562 cells. a Immunoblotting of cell lysates with anti-GFP shows bands compatible with the expected molecular weights for GFP (27 kDa) and mature PCDHB11-GFP (110 kDa); image representative of three transfections. b Immunoblotting of cell lysates with anti-HA shows bands compatible with the expected molecular weights for mature HA-PCDHB11-GFP (113 kDa) and HA-PCDHGA3 (99 kDa); image representative of two transfections. c Proportion of medium and large clusters was lower in HA-PCDHB11-GFP-transfected than in HA-PCDHGA3-transfected cells, but higher than in control cells ( n = 10-12 images per condition). d Proportion of medium to large cell clusters when normalized by HA-PCDHGA3. For visualization of surface protocadherins, live cells transfected with GFP alone (e) or in combination with HA-PCDHB11-GFP (f) or HA-PCDHGA3 (g) were stained with anti-HA-biotin and <t>streptavidin-Alexa555,</t> then fixed. Blue: DAPI. Green: GFP. Red: surface HA-tagged protocadherins. Scale bar: 10 μm
    Streptavidin Horseradish Peroxidase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 229 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Functional test of PCDHB11, the most human-specific neuronal surface protein"

    Article Title: Functional test of PCDHB11, the most human-specific neuronal surface protein

    Journal: BMC Evolutionary Biology

    doi: 10.1186/s12862-016-0652-x

    Expression of protocadherins. GFP was electroporated alone or in combination with protocadherins into K562 cells. a Immunoblotting of cell lysates with anti-GFP shows bands compatible with the expected molecular weights for GFP (27 kDa) and mature PCDHB11-GFP (110 kDa); image representative of three transfections. b Immunoblotting of cell lysates with anti-HA shows bands compatible with the expected molecular weights for mature HA-PCDHB11-GFP (113 kDa) and HA-PCDHGA3 (99 kDa); image representative of two transfections. c Proportion of medium and large clusters was lower in HA-PCDHB11-GFP-transfected than in HA-PCDHGA3-transfected cells, but higher than in control cells ( n = 10-12 images per condition). d Proportion of medium to large cell clusters when normalized by HA-PCDHGA3. For visualization of surface protocadherins, live cells transfected with GFP alone (e) or in combination with HA-PCDHB11-GFP (f) or HA-PCDHGA3 (g) were stained with anti-HA-biotin and streptavidin-Alexa555, then fixed. Blue: DAPI. Green: GFP. Red: surface HA-tagged protocadherins. Scale bar: 10 μm
    Figure Legend Snippet: Expression of protocadherins. GFP was electroporated alone or in combination with protocadherins into K562 cells. a Immunoblotting of cell lysates with anti-GFP shows bands compatible with the expected molecular weights for GFP (27 kDa) and mature PCDHB11-GFP (110 kDa); image representative of three transfections. b Immunoblotting of cell lysates with anti-HA shows bands compatible with the expected molecular weights for mature HA-PCDHB11-GFP (113 kDa) and HA-PCDHGA3 (99 kDa); image representative of two transfections. c Proportion of medium and large clusters was lower in HA-PCDHB11-GFP-transfected than in HA-PCDHGA3-transfected cells, but higher than in control cells ( n = 10-12 images per condition). d Proportion of medium to large cell clusters when normalized by HA-PCDHGA3. For visualization of surface protocadherins, live cells transfected with GFP alone (e) or in combination with HA-PCDHB11-GFP (f) or HA-PCDHGA3 (g) were stained with anti-HA-biotin and streptavidin-Alexa555, then fixed. Blue: DAPI. Green: GFP. Red: surface HA-tagged protocadherins. Scale bar: 10 μm

    Techniques Used: Expressing, Transfection, Staining

    2) Product Images from "Nuclear envelope-associated endosomes deliver surface proteins to the nucleus"

    Article Title: Nuclear envelope-associated endosomes deliver surface proteins to the nucleus

    Journal: Nature Communications

    doi: 10.1038/ncomms9218

    PE localizes in the nuclear envelope lumen and nucleoplasm. ( a ) MG63 cells intoxicated for 1 h with biotinylated PE and stained with gold-labelled streptavidin (scale bar, 100 nm). ( b ) Schematic of image shown in a with gold particles indicated by arrows. ( c ) Number of gold particles per cell in different compartments. ( d ) Number of gold particles per μm 2 in the cytosol and nucleus. Representative of two independent experiments (error bar indicates s.d.). ( e ) Fractionation of cells intoxicated with biotinylated PE for 1 h with (right panel) or without (left panel) BFA pre-treatment (C, cytosolic fraction; M, membrane fraction; N, nuclear fraction). ( f , g ) Nu-T construct and its nuclear localization (scale bar, 25 μm). ( h ) Biotinylated PE detection after pulldown of Nu-T ( Supplementary Fig. 5a,b ). ( i ) Kinetics of PE pulldown. ( e , h , i ) Representative of at least five independent experiments. Cy, cytosol; GA, Golgi apparatus; NE, nuclear envelope; Nu, nucleus; MT, mitochondria; V, vesicles.
    Figure Legend Snippet: PE localizes in the nuclear envelope lumen and nucleoplasm. ( a ) MG63 cells intoxicated for 1 h with biotinylated PE and stained with gold-labelled streptavidin (scale bar, 100 nm). ( b ) Schematic of image shown in a with gold particles indicated by arrows. ( c ) Number of gold particles per cell in different compartments. ( d ) Number of gold particles per μm 2 in the cytosol and nucleus. Representative of two independent experiments (error bar indicates s.d.). ( e ) Fractionation of cells intoxicated with biotinylated PE for 1 h with (right panel) or without (left panel) BFA pre-treatment (C, cytosolic fraction; M, membrane fraction; N, nuclear fraction). ( f , g ) Nu-T construct and its nuclear localization (scale bar, 25 μm). ( h ) Biotinylated PE detection after pulldown of Nu-T ( Supplementary Fig. 5a,b ). ( i ) Kinetics of PE pulldown. ( e , h , i ) Representative of at least five independent experiments. Cy, cytosol; GA, Golgi apparatus; NE, nuclear envelope; Nu, nucleus; MT, mitochondria; V, vesicles.

    Techniques Used: Staining, Fractionation, Construct

    3) Product Images from "Oligomeric Structure of Type I and Type II Transforming Growth Factor ? Receptors: Homodimers Form in the ER and Persist at the Plasma Membrane "

    Article Title: Oligomeric Structure of Type I and Type II Transforming Growth Factor ? Receptors: Homodimers Form in the ER and Persist at the Plasma Membrane

    Journal: The Journal of Cell Biology

    doi:

    Coimmunoprecipitation of TβRI-myc with TβRI-HA. COS7 cells were transiently transfected with TβRI-myc ( IM ), TβRI-HA ( IH ), or both ( IM + IH ), as indicated at the bottom of each lane. Immunoprecipitation was either with α-myc or α-HA, as indicated at the top of the gel. Blotting was always with α-myc, followed by biotinylated GαM, streptavidin–horseradish peroxidase, and ECL. All lanes were loaded with immunoprecipitates derived from the same amounts of cell lysates (three 10-cm dishes), except where both immunoprecipitation and blotting were with α-myc (1/3 of the above lysate in A , lane 1 ; 1/18 in A , lane 2 and in B , lane 1 ). ( A ) Effects of ligand and/or DTT treatment. The gels were run under reducing conditions. In lanes 6 and 8 , the cells were preincubated with 250 pM TGF-β1 (2 h, 4°C). In lanes 7 and 8 , the cells were pre-treated with DTT (see Materials and Methods). TβRI appears in two bands, around 59 and 53 kD. The nonspecific band at the bottom of lanes 3–8 is a component of the α-HA antibodies used for the immunoprecipitation in these lanes (see text). ( B ) Effect of N -glycosidase F. The 59-kD band collapses to 53 kD, indicating that it represents N -glycosylated receptors. The immunoprecipitates were treated with N -glycosidase F as described under Materials and Methods. The gel was run under nonreducing conditions. In lane 3 , the cells were preincubated with 250 pM TGF-β1. The band seen at 62 kD under these conditions is a nonspecific band. (The specific 59-kD band disappears).
    Figure Legend Snippet: Coimmunoprecipitation of TβRI-myc with TβRI-HA. COS7 cells were transiently transfected with TβRI-myc ( IM ), TβRI-HA ( IH ), or both ( IM + IH ), as indicated at the bottom of each lane. Immunoprecipitation was either with α-myc or α-HA, as indicated at the top of the gel. Blotting was always with α-myc, followed by biotinylated GαM, streptavidin–horseradish peroxidase, and ECL. All lanes were loaded with immunoprecipitates derived from the same amounts of cell lysates (three 10-cm dishes), except where both immunoprecipitation and blotting were with α-myc (1/3 of the above lysate in A , lane 1 ; 1/18 in A , lane 2 and in B , lane 1 ). ( A ) Effects of ligand and/or DTT treatment. The gels were run under reducing conditions. In lanes 6 and 8 , the cells were preincubated with 250 pM TGF-β1 (2 h, 4°C). In lanes 7 and 8 , the cells were pre-treated with DTT (see Materials and Methods). TβRI appears in two bands, around 59 and 53 kD. The nonspecific band at the bottom of lanes 3–8 is a component of the α-HA antibodies used for the immunoprecipitation in these lanes (see text). ( B ) Effect of N -glycosidase F. The 59-kD band collapses to 53 kD, indicating that it represents N -glycosylated receptors. The immunoprecipitates were treated with N -glycosidase F as described under Materials and Methods. The gel was run under nonreducing conditions. In lane 3 , the cells were preincubated with 250 pM TGF-β1. The band seen at 62 kD under these conditions is a nonspecific band. (The specific 59-kD band disappears).

    Techniques Used: Transfection, Immunoprecipitation, Derivative Assay

    4) Product Images from "CD8? Endows CD8 with Efficient Coreceptor Function by Coupling T Cell Receptor/CD3 to Raft-associated CD8/p56lck Complexes"

    Article Title: CD8? Endows CD8 with Efficient Coreceptor Function by Coupling T Cell Receptor/CD3 to Raft-associated CD8/p56lck Complexes

    Journal: The Journal of Experimental Medicine

    doi:

    Palmitoylation of CD8β is essential for CD8 localization in rafts and efficient lck association. (A) T1.4 hybridomas with the indicated CD8 expression were surface-biotinylated, lysed in cold TX-100 (1%), and fractionated in M and DIM fractions. These were immunoprecipitated with anti-CD8α mAb 53.6.72. The samples were analyzed by SDS-PAGE and Western blotting with streptavidin. (B–E) T1.4 hybridomas with the indicated CD8 expression were lysed in Brij96 (1%) and the lysates immunoprecipitated with mAb 53.6.72 (B and D) or anti-CD8β mAB H35–17 (C and E). The immunoprecipitates were analyzed by SDS-PAGE and Western blotting with anti-lck mAb 3A5 (B and C) or anti-CD8α antiserum (D and E). Note that anti-CD8β mAb H35 is unable to precipitate CD8αα and CD8αα β . One out of two experiments is shown.
    Figure Legend Snippet: Palmitoylation of CD8β is essential for CD8 localization in rafts and efficient lck association. (A) T1.4 hybridomas with the indicated CD8 expression were surface-biotinylated, lysed in cold TX-100 (1%), and fractionated in M and DIM fractions. These were immunoprecipitated with anti-CD8α mAb 53.6.72. The samples were analyzed by SDS-PAGE and Western blotting with streptavidin. (B–E) T1.4 hybridomas with the indicated CD8 expression were lysed in Brij96 (1%) and the lysates immunoprecipitated with mAb 53.6.72 (B and D) or anti-CD8β mAB H35–17 (C and E). The immunoprecipitates were analyzed by SDS-PAGE and Western blotting with anti-lck mAb 3A5 (B and C) or anti-CD8α antiserum (D and E). Note that anti-CD8β mAb H35 is unable to precipitate CD8αα and CD8αα β . One out of two experiments is shown.

    Techniques Used: Expressing, Immunoprecipitation, SDS Page, Western Blot

    5) Product Images from "F5-peptide induces aspermatogenesis by disrupting organization of actin- and microtubule-based cytoskeletons in the testis"

    Article Title: F5-peptide induces aspermatogenesis by disrupting organization of actin- and microtubule-based cytoskeletons in the testis

    Journal: Oncotarget

    doi: 10.18632/oncotarget.11887

    Overexpression of F5-peptide in the test is perturbs BTB function in vivo BTB integrity assay was performed to assess the ability of an intact BTB to block the diffusion of a small molecule biotin (EZ-Link Sulfo-NHS-LC-Biotin, Mr 556.59) across the BTB which biotinylated proteins in the adluminal compartment. In normal rat testes and testes transfected with pCI-neo alone (control), the functional BTB blocked biotin reagent from entering the adluminal compartment so that only proteins at the BTB and the interstitial space were labeled. Biotinylated proteins were subsequently visualized using frozen sections of the testis and stained with Alexa Fluor 488-streptavidin (green fluorescence). Distance traveled by biotin beyond the basement membrane/tunica propria was annotated by a yellow bracket. In control testes, biotin failed to travel beyond the basement membrane/tunica propria which was annotated by a dashed white line. In testes from rats treated with CdCl 2 (positive control) or transfected with pCI-neo/F5, biotin penetrated well into the adluminal compartment, illustrating that F5-peptide perturbed the BTB integrity. Lower magnified images of ~3–4 tubules were shown in insets and the enlarged image of a typical tubule was shown in the micrograph. Scale bars, 50 μm, and 200 μm in insets, which applies to corresponding micrographs and/or insets. Histograms show the semi-quantitative data by comparing the distance of biotin traveled into the epithelium (D Biotin ) vs. the radius of seminiferous tubule (D Radius ). For oblique sections, the radius of the tubule was obtained by averaging the longest and shortest distance from the basement membrane. Each bar is a mean ± SD of ~50 tubules that were randomly selected and scored from testes of 6 rats with a total of 300 randomly selected tubules. ** P
    Figure Legend Snippet: Overexpression of F5-peptide in the test is perturbs BTB function in vivo BTB integrity assay was performed to assess the ability of an intact BTB to block the diffusion of a small molecule biotin (EZ-Link Sulfo-NHS-LC-Biotin, Mr 556.59) across the BTB which biotinylated proteins in the adluminal compartment. In normal rat testes and testes transfected with pCI-neo alone (control), the functional BTB blocked biotin reagent from entering the adluminal compartment so that only proteins at the BTB and the interstitial space were labeled. Biotinylated proteins were subsequently visualized using frozen sections of the testis and stained with Alexa Fluor 488-streptavidin (green fluorescence). Distance traveled by biotin beyond the basement membrane/tunica propria was annotated by a yellow bracket. In control testes, biotin failed to travel beyond the basement membrane/tunica propria which was annotated by a dashed white line. In testes from rats treated with CdCl 2 (positive control) or transfected with pCI-neo/F5, biotin penetrated well into the adluminal compartment, illustrating that F5-peptide perturbed the BTB integrity. Lower magnified images of ~3–4 tubules were shown in insets and the enlarged image of a typical tubule was shown in the micrograph. Scale bars, 50 μm, and 200 μm in insets, which applies to corresponding micrographs and/or insets. Histograms show the semi-quantitative data by comparing the distance of biotin traveled into the epithelium (D Biotin ) vs. the radius of seminiferous tubule (D Radius ). For oblique sections, the radius of the tubule was obtained by averaging the longest and shortest distance from the basement membrane. Each bar is a mean ± SD of ~50 tubules that were randomly selected and scored from testes of 6 rats with a total of 300 randomly selected tubules. ** P

    Techniques Used: Over Expression, In Vivo, Integrity Assay, Blocking Assay, Diffusion-based Assay, Transfection, Functional Assay, Labeling, Staining, Fluorescence, Positive Control

    6) Product Images from "Secretory Immunoglobulin A Antibodies against the ?1 Outer Capsid Protein of Reovirus Type 1 Lang Prevent Infection of Mouse Peyer's Patches †"

    Article Title: Secretory Immunoglobulin A Antibodies against the ?1 Outer Capsid Protein of Reovirus Type 1 Lang Prevent Infection of Mouse Peyer's Patches †

    Journal: Journal of Virology

    doi: 10.1128/JVI.78.2.947-957.2004

    Binding of antireovirus MAbs to whole reovirus T1L or recoated T1L viral cores lacking σ1. Reovirus T1L particles (A) or T1L cores recoated with σ3 and μ1 but lacking σ1 (B) were dotted onto nitrocellulose paper and incubated with hybridoma supernatants (rows 1 to 3), mouse fecal extract (row 4), or buffer (rows 5 and 6) followed by biotinylated anti-IgA or -IgG secondary antibodies and HRP-streptavidin. Row 1, antireovirus IgA MAb 2F3 (shown here) and five other IgA MAbs (data not shown) failed to recognize either whole virus or recoated cores. Row 2, IgA MAb 1E1 recognized whole virus but failed to recognize recoated cores lacking σ1. Row 3, IgG MAb 5C6, specific for the T1L σ1 protein, recognized whole virus but not recoated cores. Row 4, pooled fecal extracts from BALB/c mice orally immunized with reovirus T1L contained IgA that recognized both whole virus and recoated cores. Rows 5 and 6, control dots exposed to anti-mouse IgA (row 5) or IgG (row 6) secondary antibodies.
    Figure Legend Snippet: Binding of antireovirus MAbs to whole reovirus T1L or recoated T1L viral cores lacking σ1. Reovirus T1L particles (A) or T1L cores recoated with σ3 and μ1 but lacking σ1 (B) were dotted onto nitrocellulose paper and incubated with hybridoma supernatants (rows 1 to 3), mouse fecal extract (row 4), or buffer (rows 5 and 6) followed by biotinylated anti-IgA or -IgG secondary antibodies and HRP-streptavidin. Row 1, antireovirus IgA MAb 2F3 (shown here) and five other IgA MAbs (data not shown) failed to recognize either whole virus or recoated cores. Row 2, IgA MAb 1E1 recognized whole virus but failed to recognize recoated cores lacking σ1. Row 3, IgG MAb 5C6, specific for the T1L σ1 protein, recognized whole virus but not recoated cores. Row 4, pooled fecal extracts from BALB/c mice orally immunized with reovirus T1L contained IgA that recognized both whole virus and recoated cores. Rows 5 and 6, control dots exposed to anti-mouse IgA (row 5) or IgG (row 6) secondary antibodies.

    Techniques Used: Binding Assay, Incubation, Mouse Assay

    7) Product Images from "Role of the polypeptide N-acetylgalactosaminyltransferase 3 in ovarian cancer progression: possible implications in abnormal mucin O-glycosylation"

    Article Title: Role of the polypeptide N-acetylgalactosaminyltransferase 3 in ovarian cancer progression: possible implications in abnormal mucin O-glycosylation

    Journal: Oncotarget

    doi:

    Analysis of GALNT3-mediated MUC1 glycosylation in EOC cells A. Western blot analysis of GALNT3 and MUC1 endogenous protein expression in different EOC cell lines, including A2780s; β-actin was used as a loading control; B. Semi-quantitative RT-PCR analysis of MUC1 mRNA levels in the control clone (ctrl) and shRNA- GALNT3 clones 1 and 2 (sh-G1 and sh-G2); the GUSB gene was used as an internal control; C. Western blot analysis of MUC1 expression in the ctrl, the sh-G1 and the sh-G2 A2780s clones before (input) and following pull-down assay using biotin-conjugated VVA lectin and streptavidin agarose (pull-down); D. VVA-lectin-mediated immunoblot analysis of GalNAc-conjugated proteins in protein lysates of the ctrl, the sh-G1 and the sh-G2 A2780s clones following VVA lectin pull-down assay (pull-down). The arrow indicates bands corresponding to possible GalNAc-conjugated MUC1 peptides; E. Western blot analysis for E-cadherin and β-catenin expression in the ctrl, the sh-G1 and the sh-G2 A2780s clones; β-actin was used as a loading control
    Figure Legend Snippet: Analysis of GALNT3-mediated MUC1 glycosylation in EOC cells A. Western blot analysis of GALNT3 and MUC1 endogenous protein expression in different EOC cell lines, including A2780s; β-actin was used as a loading control; B. Semi-quantitative RT-PCR analysis of MUC1 mRNA levels in the control clone (ctrl) and shRNA- GALNT3 clones 1 and 2 (sh-G1 and sh-G2); the GUSB gene was used as an internal control; C. Western blot analysis of MUC1 expression in the ctrl, the sh-G1 and the sh-G2 A2780s clones before (input) and following pull-down assay using biotin-conjugated VVA lectin and streptavidin agarose (pull-down); D. VVA-lectin-mediated immunoblot analysis of GalNAc-conjugated proteins in protein lysates of the ctrl, the sh-G1 and the sh-G2 A2780s clones following VVA lectin pull-down assay (pull-down). The arrow indicates bands corresponding to possible GalNAc-conjugated MUC1 peptides; E. Western blot analysis for E-cadherin and β-catenin expression in the ctrl, the sh-G1 and the sh-G2 A2780s clones; β-actin was used as a loading control

    Techniques Used: Western Blot, Expressing, Quantitative RT-PCR, shRNA, Clone Assay, Pull Down Assay

    8) Product Images from "An improved method for the in vitro evolution of aptamers and applications in protein detection and purification"

    Article Title: An improved method for the in vitro evolution of aptamers and applications in protein detection and purification

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gng110

    SDS–PAGE analysis of aptamer affinity purification of TTF1 protein from E.coli lysates using biotinylated TTF1 aptamer ‘A’ immobilized on streptavidin magnetic beads. The gel is 4–20% polyacrylamide stained with GelCode blue. Lane 1 contains cleared lysate from E.coli expressing the protein of interest, and lane 2 contains the cleared lysate spiked with Ni-NTA-purified TTF1 protein (lane 3). Material in lane 4 was from 10 min binding and 2 h elution at 4°C. Material in lane 6 was from 30 min binding and 15 min elution at room temperature. Material in lane 8 was from 5 min binding and 5 min elution at room temperature. After each elution with Benzonase, any remaining protein was removed from the aptamer with 0.1% SDS (lanes 5, 7 and 9).
    Figure Legend Snippet: SDS–PAGE analysis of aptamer affinity purification of TTF1 protein from E.coli lysates using biotinylated TTF1 aptamer ‘A’ immobilized on streptavidin magnetic beads. The gel is 4–20% polyacrylamide stained with GelCode blue. Lane 1 contains cleared lysate from E.coli expressing the protein of interest, and lane 2 contains the cleared lysate spiked with Ni-NTA-purified TTF1 protein (lane 3). Material in lane 4 was from 10 min binding and 2 h elution at 4°C. Material in lane 6 was from 30 min binding and 15 min elution at room temperature. Material in lane 8 was from 5 min binding and 5 min elution at room temperature. After each elution with Benzonase, any remaining protein was removed from the aptamer with 0.1% SDS (lanes 5, 7 and 9).

    Techniques Used: SDS Page, Affinity Purification, Magnetic Beads, Staining, Expressing, Purification, Binding Assay

    Comparison of the specificity of TTF1 aptamer ‘A’ to a monoclonal anti-PentaHis antibody in a protein blot analysis. Lane 1 contains cleared lysate from E.coli expressing the HOX4 homeodomain. Lane 2 contains purified HOX4 homeodomain protein (marked with an arrow). Lane 3 contains cleared lysate from E.coli expressing TTF1. Lane 4 contains purified TTF1 protein (marked with an arrow). ( A ) 4–20% SDS–PAGE stained with GelCode blue. ( B ) Blot of the material shown in (A) probed with an anti-PentaHis monoclonal antibody. ( C ) Blot of the material shown in (A) probed with the biotinylated TTF1 aptamer ‘A’. Note that the lower dark bands in lanes 1 and 3 of (C) were detected by the secondary probe, streptavidin–HRP (not shown).
    Figure Legend Snippet: Comparison of the specificity of TTF1 aptamer ‘A’ to a monoclonal anti-PentaHis antibody in a protein blot analysis. Lane 1 contains cleared lysate from E.coli expressing the HOX4 homeodomain. Lane 2 contains purified HOX4 homeodomain protein (marked with an arrow). Lane 3 contains cleared lysate from E.coli expressing TTF1. Lane 4 contains purified TTF1 protein (marked with an arrow). ( A ) 4–20% SDS–PAGE stained with GelCode blue. ( B ) Blot of the material shown in (A) probed with an anti-PentaHis monoclonal antibody. ( C ) Blot of the material shown in (A) probed with the biotinylated TTF1 aptamer ‘A’. Note that the lower dark bands in lanes 1 and 3 of (C) were detected by the secondary probe, streptavidin–HRP (not shown).

    Techniques Used: Expressing, Purification, SDS Page, Staining

    9) Product Images from "Detection of a Sulfotransferase (HEC-GlcNAc6ST) in High Endothelial Venules of Lymph Nodes and in High Endothelial Venule-Like Vessels within Ectopic Lymphoid Aggregates "

    Article Title: Detection of a Sulfotransferase (HEC-GlcNAc6ST) in High Endothelial Venules of Lymph Nodes and in High Endothelial Venule-Like Vessels within Ectopic Lymphoid Aggregates

    Journal: The American Journal of Pathology

    doi:

    Contribution of HEC-GlcNAC6ST to the generation of luminal and abluminal MECA-79 epitopes. Cryostat sections of PN were prepared from wild-type ( A , C ) and −/− ( B , D ) mice that had received intravenous injections of unlabeled MECA-79. The sections were stained first with Cy3-conjugated secondary reagents to detect injected luminally bound MECA-79 and then with biotinylated MECA-79 followed by streptavidin-Cy2 to detect abluminal MECA-79 epitopes. C and D: Overlay of the two fluorescent images. A and B show the corresponding bright-field images. Scale bar, 25 μm.
    Figure Legend Snippet: Contribution of HEC-GlcNAC6ST to the generation of luminal and abluminal MECA-79 epitopes. Cryostat sections of PN were prepared from wild-type ( A , C ) and −/− ( B , D ) mice that had received intravenous injections of unlabeled MECA-79. The sections were stained first with Cy3-conjugated secondary reagents to detect injected luminally bound MECA-79 and then with biotinylated MECA-79 followed by streptavidin-Cy2 to detect abluminal MECA-79 epitopes. C and D: Overlay of the two fluorescent images. A and B show the corresponding bright-field images. Scale bar, 25 μm.

    Techniques Used: Mouse Assay, Staining, Injection

    10) Product Images from "Heterogeneous Nuclear Ribonucleoprotein (hnRNP) E1 Binds to hnRNP A2 and Inhibits Translation of A2 Response Element mRNAs"

    Article Title: Heterogeneous Nuclear Ribonucleoprotein (hnRNP) E1 Binds to hnRNP A2 and Inhibits Translation of A2 Response Element mRNAs

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E05-10-0946

    hnRNP E1–hnRNP A2 interaction. (A) Coimmunoprecipitation: in vitro translation products. In vitro synthesized [ 35 S]methionine-labeled hnRNP A2 was combined with a fivefold molar excess of hnRNP A2-HA or hnRNP E1-25-HA, and the mixture was precipitated with anti-HA antibodies. The precipitates were separated by SDS-PAGE; the gel was fixed, dried, and exposed to x-ray film. (B) Coimmunoprecipitation: in vivo endogenous products. Oligodendrocyte homogenate was incubated with rabbit anti-hnRNP E1 or with normal rabbit serum (NRS). The immunoprecipitates were isolated with protein A-conjugated agarose beads, separated by SDS-PAGE, and subjected to Western blot analysis for the presence of hnRNP A2 using mouse anti-hnRNP A2. The right lane is an aliquot of the homogenate before immunoprecipitation. (C) Biotin transfer assay: hnRNP A2 (A2) conjugated with Sulfo-SBED was mixed with hnRNP E1 (E1), with BSA, or without any addition, and subjected (+) or not (−) to UV cross-linking. After reduction, the protein mixtures were analyzed by SDS-PAGE, blotted onto PVDF membrane, and probed with streptavidin-horseradish peroxidase (top). Corresponding portions were analyzed by SDS-PAGE and stained with Coomassie blue (bottom). Molecular weight markers are displayed to the left.
    Figure Legend Snippet: hnRNP E1–hnRNP A2 interaction. (A) Coimmunoprecipitation: in vitro translation products. In vitro synthesized [ 35 S]methionine-labeled hnRNP A2 was combined with a fivefold molar excess of hnRNP A2-HA or hnRNP E1-25-HA, and the mixture was precipitated with anti-HA antibodies. The precipitates were separated by SDS-PAGE; the gel was fixed, dried, and exposed to x-ray film. (B) Coimmunoprecipitation: in vivo endogenous products. Oligodendrocyte homogenate was incubated with rabbit anti-hnRNP E1 or with normal rabbit serum (NRS). The immunoprecipitates were isolated with protein A-conjugated agarose beads, separated by SDS-PAGE, and subjected to Western blot analysis for the presence of hnRNP A2 using mouse anti-hnRNP A2. The right lane is an aliquot of the homogenate before immunoprecipitation. (C) Biotin transfer assay: hnRNP A2 (A2) conjugated with Sulfo-SBED was mixed with hnRNP E1 (E1), with BSA, or without any addition, and subjected (+) or not (−) to UV cross-linking. After reduction, the protein mixtures were analyzed by SDS-PAGE, blotted onto PVDF membrane, and probed with streptavidin-horseradish peroxidase (top). Corresponding portions were analyzed by SDS-PAGE and stained with Coomassie blue (bottom). Molecular weight markers are displayed to the left.

    Techniques Used: In Vitro, Synthesized, Labeling, SDS Page, In Vivo, Incubation, Isolation, Western Blot, Immunoprecipitation, Staining, Molecular Weight

    11) Product Images from "Carbohydrate Mimetic Peptides Augment Carbohydrate-Reactive Immune Responses in the Absence of Immune Pathology"

    Article Title: Carbohydrate Mimetic Peptides Augment Carbohydrate-Reactive Immune Responses in the Absence of Immune Pathology

    Journal: Cancers

    doi: 10.3390/cancers3044151

    Serum IgM antibodies and GS-I binding to 4T1 cell lysate. Whole-cell lysate from 4T1 cell line was prepared. Mice (5/group) were pre-bled and immunized with peptide 107 on days 0 and 14. Sera were collected 7 days after the peptide boost and pooled for each group. Western blot analysis: Sera were diluted 1:1000 for the western. Anti mouse IgM used as secondary antibody. Binding with biotin-conjugated GS-I was followed by streptavidin-HRP.
    Figure Legend Snippet: Serum IgM antibodies and GS-I binding to 4T1 cell lysate. Whole-cell lysate from 4T1 cell line was prepared. Mice (5/group) were pre-bled and immunized with peptide 107 on days 0 and 14. Sera were collected 7 days after the peptide boost and pooled for each group. Western blot analysis: Sera were diluted 1:1000 for the western. Anti mouse IgM used as secondary antibody. Binding with biotin-conjugated GS-I was followed by streptavidin-HRP.

    Techniques Used: Binding Assay, Mouse Assay, Western Blot

    12) Product Images from "Demonstration of Array-Based Analysis for Highly Multiplexed PCR Assays"

    Article Title: Demonstration of Array-Based Analysis for Highly Multiplexed PCR Assays

    Journal: The Journal of Molecular Diagnostics : JMD

    doi: 10.1016/j.jmoldx.2010.11.019

    Representative MPAD assay. PCR products are hybridized to the LDA containing the designated BCL2 features, placed in duplicate. After high-stringency washes, the LDAs are incubated with streptavidin–horseradish peroxidase to label products containing
    Figure Legend Snippet: Representative MPAD assay. PCR products are hybridized to the LDA containing the designated BCL2 features, placed in duplicate. After high-stringency washes, the LDAs are incubated with streptavidin–horseradish peroxidase to label products containing

    Techniques Used: Polymerase Chain Reaction, Incubation

    13) Product Images from "Na+/H+ exchanger type 1 is a receptor for pathogenic subgroup J avian leukosis virus"

    Article Title: Na+/H+ exchanger type 1 is a receptor for pathogenic subgroup J avian leukosis virus

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0509785103

    A 90-kDa chicken protein associates with the SU domain of EnvJ. ( A ) QT6 or DF-1 cells were incubated with SARS S1-Ig (shaded area, negative control), SUA-Ig (gray line), or SUJ-Ig (black line) and then a Cy5-conjugated goat anti-rabbit secondary antibody. Binding of immunoadhesins to cells was measured by flow cytometry. ( B ) Surface-biotinylated QT6 or DF-1 cell lysates were precipitated with SUJ-Ig or SUA-Ig. Precipitated proteins were subjected to SDS/PAGE and Western blot analysis by using streptavidin horseradish peroxidase. ( C ) QT6 or DF-1 cell lysates were precipitated with SUJ-Ig or SUA-Ig. Precipitated proteins were subjected to SDS/PAGE and Western blot analysis by using an anti-NHE1 monoclonal antibody. FL4-H denotes fluorescence intensity.
    Figure Legend Snippet: A 90-kDa chicken protein associates with the SU domain of EnvJ. ( A ) QT6 or DF-1 cells were incubated with SARS S1-Ig (shaded area, negative control), SUA-Ig (gray line), or SUJ-Ig (black line) and then a Cy5-conjugated goat anti-rabbit secondary antibody. Binding of immunoadhesins to cells was measured by flow cytometry. ( B ) Surface-biotinylated QT6 or DF-1 cell lysates were precipitated with SUJ-Ig or SUA-Ig. Precipitated proteins were subjected to SDS/PAGE and Western blot analysis by using streptavidin horseradish peroxidase. ( C ) QT6 or DF-1 cell lysates were precipitated with SUJ-Ig or SUA-Ig. Precipitated proteins were subjected to SDS/PAGE and Western blot analysis by using an anti-NHE1 monoclonal antibody. FL4-H denotes fluorescence intensity.

    Techniques Used: Incubation, Negative Control, Binding Assay, Flow Cytometry, Cytometry, SDS Page, Western Blot, Fluorescence

    14) Product Images from "Separation of Insulin Signaling into Distinct GLUT4 Translocation and Activation Steps"

    Article Title: Separation of Insulin Signaling into Distinct GLUT4 Translocation and Activation Steps

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.24.17.7567-7577.2004

    PBP10 did not inhibit IRAP endocytosis. 3T3-L1 adipocytes were either left untreated (lane a) or treated with 100 nM insulin (lanes b and c). Insulin was removed, and cell surface proteins were biotinylated as described in Materials and Methods. The cells were incubated for 5 min at 37°C in the absence (lanes a and b) or presence (lane c) of PBP10 to allow endocytosis, chilled again, and incubated with a cell-impermeable biotin cleavage solution. Then, the cells were lysed, immunoprecipitated with anti-IRAP antibody, and immunoblotted either with streptavidin-horseradish peroxidase (top) or with anti-IRAP antibody (bottom). A representative result of three independent experiments is shown.
    Figure Legend Snippet: PBP10 did not inhibit IRAP endocytosis. 3T3-L1 adipocytes were either left untreated (lane a) or treated with 100 nM insulin (lanes b and c). Insulin was removed, and cell surface proteins were biotinylated as described in Materials and Methods. The cells were incubated for 5 min at 37°C in the absence (lanes a and b) or presence (lane c) of PBP10 to allow endocytosis, chilled again, and incubated with a cell-impermeable biotin cleavage solution. Then, the cells were lysed, immunoprecipitated with anti-IRAP antibody, and immunoblotted either with streptavidin-horseradish peroxidase (top) or with anti-IRAP antibody (bottom). A representative result of three independent experiments is shown.

    Techniques Used: Incubation, Immunoprecipitation

    15) Product Images from "Cytotoxic T Lymphocyte Antigen 4 Is Induced in the Thymus upon In Vivo Activation and Its Blockade Prevents Anti-CD3-mediated Depletion of Thymocytes "

    Article Title: Cytotoxic T Lymphocyte Antigen 4 Is Induced in the Thymus upon In Vivo Activation and Its Blockade Prevents Anti-CD3-mediated Depletion of Thymocytes

    Journal: The Journal of Experimental Medicine

    doi:

    CTLA-4 expression is induced upon in vivo anti-CD3 administration. Mice were injected intraperitoneally with 100 μg of anti-CD3 mAbs or with saline as control. After 24 h mice were killed and thymocytes were analyzed for CTLA-4 expression by three-color flow cytometry. ( A ) Intracellular expression of CTLA-4: thymocytes were first double stained with anti-CD4–FITC and anti-CD8–biotin surface markers, fixed, permeabilized, and finally incubated with anti–CTLA-4–PE. ( B ) Cell surface expression of CTLA-4: thymocytes were triple stained with anti-CD4–FITC, anti-CD8–biotin, and anti–CTLA-4–PE. In all experiments biotin has been revealed by incubation with streptavidin Cy-Chrome. Contour dot-plots represent CTLA-4 expression evaluated on viable thymocyte subsets by differential gating based on CD4 and CD8 expression ( boxes ). These results are representative of at least three independent experiments.
    Figure Legend Snippet: CTLA-4 expression is induced upon in vivo anti-CD3 administration. Mice were injected intraperitoneally with 100 μg of anti-CD3 mAbs or with saline as control. After 24 h mice were killed and thymocytes were analyzed for CTLA-4 expression by three-color flow cytometry. ( A ) Intracellular expression of CTLA-4: thymocytes were first double stained with anti-CD4–FITC and anti-CD8–biotin surface markers, fixed, permeabilized, and finally incubated with anti–CTLA-4–PE. ( B ) Cell surface expression of CTLA-4: thymocytes were triple stained with anti-CD4–FITC, anti-CD8–biotin, and anti–CTLA-4–PE. In all experiments biotin has been revealed by incubation with streptavidin Cy-Chrome. Contour dot-plots represent CTLA-4 expression evaluated on viable thymocyte subsets by differential gating based on CD4 and CD8 expression ( boxes ). These results are representative of at least three independent experiments.

    Techniques Used: Expressing, In Vivo, Mouse Assay, Injection, Flow Cytometry, Cytometry, Staining, Incubation

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    Autoradiography:

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    Enzyme-linked Immunosorbent Assay:

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    Incubation:

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    Western Blot:

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    Immunoprecipitation:

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    Cell Culture:

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    Polymerase Chain Reaction:

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    Recombinant:

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    Nucleic Acid Electrophoresis:

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    Isolation:

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    Article Snippet: Fusion products were seeded in 96-well flat-bottom plates with a feeder layer of thymocytes isolated from DBA/2 mice. .. Bound antibody was detected with streptavidin-horseradish peroxidase (HRP; Pierce) diluted 1:1,000, using a one-component peroxidase substrate system (Kirkegaard & Perry Laboratories, Gaithersburg, Md.).

    Article Title: CD8? Endows CD8 with Efficient Coreceptor Function by Coupling T Cell Receptor/CD3 to Raft-associated CD8/p56lck Complexes
    Article Snippet: Paragraph title: Isolation of DIM. ... The immunoprecipitates were resolved on SDS-PAGE (10%, reducing), Western blotted with streptavidin-horseradish peroxidase (HRP; GIBCO BRL) or anti-CD8α antiserum and revealed by enhanced chemoluminescence (ECL) as described ( , ).

    Transfection:

    Article Title: Na+/H+ exchanger type 1 is a receptor for pathogenic subgroup J avian leukosis virus
    Article Snippet: The resulting Western blot was probed with streptavidin–horseradish peroxidase (Invitrogen). .. 293T or QT6 cells were transfected with pCAGGS, pCAGGS-chNHE1, pCAGGS-huNHE1, pCAGGS-chNHE1AU1, or pCAGGS-huNHE1AU1 by using calcium phosphate precipitation and lysed in Brij lysis buffer 48 h after transfection.

    Article Title: Detection of a Sulfotransferase (HEC-GlcNAc6ST) in High Endothelial Venules of Lymph Nodes and in High Endothelial Venule-Like Vessels within Ectopic Lymphoid Aggregates
    Article Snippet: Three days after transfection, lysates were prepared from the transfected cells by lysis in PBS/2% Triton X-100. .. The membrane was incubated in blotto containing 1 μg/ml of purified HEC-GlcNAc6ST antibody for 1 hour at room temperature, followed by biotinylated goat anti-rabbit IgG (1 μg/ml, Jackson Immuno-Research) and streptavidin-horseradish peroxidase (Caltag Laboratories).

    Article Title: Functional test of PCDHB11, the most human-specific neuronal surface protein
    Article Snippet: For immunoblotting, transfected cells were centrifuged 10 min at 200 g, 37 °C, and the supernatants were resuspended in radioimmunoprecipitation assay (RIPA) buffer including protease inhibitors (Thermo Fisher Scientific, Waltham, MA). .. For GFP detection, membranes were incubated with a rabbit polyclonal antibody (Life Technologies, A-11122) diluted 1:2000 in 50 mM Tris, 100 mM NaCl, 0.1 % Tween 20, pH 7.4, with 3 % bovine serum albumine (Sigma, St. Louis, MO) and a horse-radish peroxidase-conjugated anti-rabbit secondary antibody (Invitrogen, Waltham, MA), while for HA revelation, they were incubated with biotinylated 3 F10 rat monoclonal antibody (Roche Life Science, Indianapolis, IN) diluted 1:500 in the same buffer, and streptavidin-horseradish peroxidase (Invitrogen, Waltham, MA).

    Microscopy:

    Article Title: Functional test of PCDHB11, the most human-specific neuronal surface protein
    Article Snippet: The plates were rocked for at least 2 h at 37 °C and 5–6 movements per minute, and 5–10 fields of view in each well were photographed on an Eclipse TE300 microscope using a 10x magnifying objective and a DS-QiMC camera (Nikon, Melville, NY). .. For GFP detection, membranes were incubated with a rabbit polyclonal antibody (Life Technologies, A-11122) diluted 1:2000 in 50 mM Tris, 100 mM NaCl, 0.1 % Tween 20, pH 7.4, with 3 % bovine serum albumine (Sigma, St. Louis, MO) and a horse-radish peroxidase-conjugated anti-rabbit secondary antibody (Invitrogen, Waltham, MA), while for HA revelation, they were incubated with biotinylated 3 F10 rat monoclonal antibody (Roche Life Science, Indianapolis, IN) diluted 1:500 in the same buffer, and streptavidin-horseradish peroxidase (Invitrogen, Waltham, MA).

    Purification:

    Article Title: An improved method for the in vitro evolution of aptamers and applications in protein detection and purification
    Article Snippet: To measure the binding of aptamers to proteins immobilized on microtiter plates, 500 ng of purified TTF1 or purified HOX4 fragment was bound to wells of an Ni-NTA HisSorb plate (Qiagen) in 200 µl of PBS-T for 2 h at room temperature. .. Streptavidin–horseradish peroxidase (HRP; Molecular Probes) was diluted 1:10 000 into PBS-T and a 200 µl aliquot was incubated with the proteins and bound aptamers in the HisSorb plate for 30 min at room temperature.

    Article Title: Detection of a Sulfotransferase (HEC-GlcNAc6ST) in High Endothelial Venules of Lymph Nodes and in High Endothelial Venule-Like Vessels within Ectopic Lymphoid Aggregates
    Article Snippet: .. The membrane was incubated in blotto containing 1 μg/ml of purified HEC-GlcNAc6ST antibody for 1 hour at room temperature, followed by biotinylated goat anti-rabbit IgG (1 μg/ml, Jackson Immuno-Research) and streptavidin-horseradish peroxidase (Caltag Laboratories). .. The blot was developed with an enhance chemiluminescence (ECL) horseradish peroxidase substrate kit (Amersham, Arlington Heights, IL) followed by exposure to X-ray film.

    Lysis:

    Article Title: Na+/H+ exchanger type 1 is a receptor for pathogenic subgroup J avian leukosis virus
    Article Snippet: Precipitates were washed twice in Brij lysis buffer and once in PBS. .. The resulting Western blot was probed with streptavidin–horseradish peroxidase (Invitrogen).

    Article Title: Oligomeric Structure of Type I and Type II Transforming Growth Factor ? Receptors: Homodimers Form in the ER and Persist at the Plasma Membrane
    Article Snippet: Immunoprecipitates were washed three times with the lysis buffer, dissolved in Laemmli loading buffer with or without mercaptoethanol, and run on 7% SDS-PAGE. .. The blot was then sequentially incubated with 20 μg/ml α-myc, 1:2,000 biotinylated GαM (goat IgG anti–mouse IgG; Jackson ImmunoResearch Laboratories ), and 1:1,000 streptavidin-horseradish peroxidase ( GIBCO-BRL ).

    Article Title: Detection of a Sulfotransferase (HEC-GlcNAc6ST) in High Endothelial Venules of Lymph Nodes and in High Endothelial Venule-Like Vessels within Ectopic Lymphoid Aggregates
    Article Snippet: Three days after transfection, lysates were prepared from the transfected cells by lysis in PBS/2% Triton X-100. .. The membrane was incubated in blotto containing 1 μg/ml of purified HEC-GlcNAc6ST antibody for 1 hour at room temperature, followed by biotinylated goat anti-rabbit IgG (1 μg/ml, Jackson Immuno-Research) and streptavidin-horseradish peroxidase (Caltag Laboratories).

    Mouse Assay:

    Article Title: Secretory Immunoglobulin A Antibodies against the ?1 Outer Capsid Protein of Reovirus Type 1 Lang Prevent Infection of Mouse Peyer's Patches †
    Article Snippet: Fusion products were seeded in 96-well flat-bottom plates with a feeder layer of thymocytes isolated from DBA/2 mice. .. Bound antibody was detected with streptavidin-horseradish peroxidase (HRP; Pierce) diluted 1:1,000, using a one-component peroxidase substrate system (Kirkegaard & Perry Laboratories, Gaithersburg, Md.).

    SDS Page:

    Article Title: Heterogeneous Nuclear Ribonucleoprotein (hnRNP) E1 Binds to hnRNP A2 and Inhibits Translation of A2 Response Element mRNAs
    Article Snippet: .. Biotin-labeled proteins were separated by SDS-PAGE and either stained with Coomassie blue or blotted onto polyvinylidene difluoride (PVDF) membrane, probed with 20 ng/ml streptavidin-horseradish peroxidase for 90 min at room temperature, and developed by enhanced chemiluminescence (Pierce Chemical) to identify biotin-conjugated proteins. ..

    Article Title: Oligomeric Structure of Type I and Type II Transforming Growth Factor ? Receptors: Homodimers Form in the ER and Persist at the Plasma Membrane
    Article Snippet: Immunoprecipitates were washed three times with the lysis buffer, dissolved in Laemmli loading buffer with or without mercaptoethanol, and run on 7% SDS-PAGE. .. The blot was then sequentially incubated with 20 μg/ml α-myc, 1:2,000 biotinylated GαM (goat IgG anti–mouse IgG; Jackson ImmunoResearch Laboratories ), and 1:1,000 streptavidin-horseradish peroxidase ( GIBCO-BRL ).

    Article Title: CD8? Endows CD8 with Efficient Coreceptor Function by Coupling T Cell Receptor/CD3 to Raft-associated CD8/p56lck Complexes
    Article Snippet: .. The immunoprecipitates were resolved on SDS-PAGE (10%, reducing), Western blotted with streptavidin-horseradish peroxidase (HRP; GIBCO BRL) or anti-CD8α antiserum and revealed by enhanced chemoluminescence (ECL) as described ( , ). ..

    Plasmid Preparation:

    Article Title: Separation of Insulin Signaling into Distinct GLUT4 Translocation and Activation Steps
    Article Snippet: Sulfo-NHS-S-S-biotin and streptavidin-horseradish peroxidase were purchased from Pierce (Rockford, Ill.). .. The plasmid to express HA-tagged GLUT4 was a generous gift from S. H. Cushman.

    Article Title: Carbohydrate Mimetic Peptides Augment Carbohydrate-Reactive Immune Responses in the Absence of Immune Pathology
    Article Snippet: Sections were then incubated with a biotinylated mixture of anti-mouse IgG and anti-mouse IgM (Dako Cytomation) for 1 hr at RT and washed in DPBS containing 0.1% Tween 20, followed by incubation with streptavidin horseradish peroxidase (1:5000, Pierce, Rockford, IL, USA) for 30 min at RT. .. To determine relative reactivity of IgG and IgM, slides were separately stained with biotinylated anti-murine IgG (Sigma, St. Louis, MO, USA, 1:400) and –IgM (Vector, Burlingame, CA, USA, 1:400) antibodies.

    Article Title: Detection of a Sulfotransferase (HEC-GlcNAc6ST) in High Endothelial Venules of Lymph Nodes and in High Endothelial Venule-Like Vessels within Ectopic Lymphoid Aggregates
    Article Snippet: cDNAs encoding murine HEC-GlcNAc6ST in pCDNA3.1/HISMyc (Invitrogen, Carlsbad, CA) or the vector control were transfected into COS cells by lipofectamine (Invitrogen)-mediated transfection. .. The membrane was incubated in blotto containing 1 μg/ml of purified HEC-GlcNAc6ST antibody for 1 hour at room temperature, followed by biotinylated goat anti-rabbit IgG (1 μg/ml, Jackson Immuno-Research) and streptavidin-horseradish peroxidase (Caltag Laboratories).

    Negative Control:

    Article Title: Carbohydrate Mimetic Peptides Augment Carbohydrate-Reactive Immune Responses in the Absence of Immune Pathology
    Article Snippet: Sections were then incubated with a biotinylated mixture of anti-mouse IgG and anti-mouse IgM (Dako Cytomation) for 1 hr at RT and washed in DPBS containing 0.1% Tween 20, followed by incubation with streptavidin horseradish peroxidase (1:5000, Pierce, Rockford, IL, USA) for 30 min at RT. .. Slides were stained using a biotinylated anti-goat antibody and with secondary antibody alone as negative control.

    Binding Assay:

    Article Title: Carbohydrate Mimetic Peptides Augment Carbohydrate-Reactive Immune Responses in the Absence of Immune Pathology
    Article Snippet: Endogenous peroxidase activity was blocked by immersion in 0.3% (w:v) hydrogen peroxide in absolute methanol (Dako Cytomation) for 10 min followed by DPBS wash. Nonspecific binding was blocked by incubating with DPBS containing 2% BSA at RT for 35 min. .. Sections were then incubated with a biotinylated mixture of anti-mouse IgG and anti-mouse IgM (Dako Cytomation) for 1 hr at RT and washed in DPBS containing 0.1% Tween 20, followed by incubation with streptavidin horseradish peroxidase (1:5000, Pierce, Rockford, IL, USA) for 30 min at RT.

    Article Title: An improved method for the in vitro evolution of aptamers and applications in protein detection and purification
    Article Snippet: To measure the binding of aptamers to proteins immobilized on microtiter plates, 500 ng of purified TTF1 or purified HOX4 fragment was bound to wells of an Ni-NTA HisSorb plate (Qiagen) in 200 µl of PBS-T for 2 h at room temperature. .. Streptavidin–horseradish peroxidase (HRP; Molecular Probes) was diluted 1:10 000 into PBS-T and a 200 µl aliquot was incubated with the proteins and bound aptamers in the HisSorb plate for 30 min at room temperature.

    Radio Immunoprecipitation:

    Article Title: Functional test of PCDHB11, the most human-specific neuronal surface protein
    Article Snippet: For immunoblotting, transfected cells were centrifuged 10 min at 200 g, 37 °C, and the supernatants were resuspended in radioimmunoprecipitation assay (RIPA) buffer including protease inhibitors (Thermo Fisher Scientific, Waltham, MA). .. For GFP detection, membranes were incubated with a rabbit polyclonal antibody (Life Technologies, A-11122) diluted 1:2000 in 50 mM Tris, 100 mM NaCl, 0.1 % Tween 20, pH 7.4, with 3 % bovine serum albumine (Sigma, St. Louis, MO) and a horse-radish peroxidase-conjugated anti-rabbit secondary antibody (Invitrogen, Waltham, MA), while for HA revelation, they were incubated with biotinylated 3 F10 rat monoclonal antibody (Roche Life Science, Indianapolis, IN) diluted 1:500 in the same buffer, and streptavidin-horseradish peroxidase (Invitrogen, Waltham, MA).

    Concentration Assay:

    Article Title: Nuclear envelope-associated endosomes deliver surface proteins to the nucleus
    Article Snippet: Cells were incubated for 60 min with PE at a final concentration of 5 μg ml−1 for HeLa cells and 3 μg ml−1 for MG63 cells. .. Membranes were incubated with actin (#3280, 1:5,000, Abcam), β-tubulin (#ab6046, 1:5,000 Abcam), EEA1 (#E7659, 1:1,000, Sigma-Aldrich), EGFR (#2232L, 1:1,000, Cell Signaling Technology), histone H1 (#sc-10806, 1:1,000, Santa Cruz), PDI (#RL77, 1:15,000, Abcam), LRP1 (#L2420, 1:1,000, Sigma-Aldrich), Rab7 (#ab137029, 1:1,000, Abcam), Sec61B (#ab78276, 1:5,000, Abcam), streptavidin-horseradish peroxidase (HRP; #21130, 1:10,000, Thermo Fisher Scientific) and SUN2 (# HPA001209, 1:2,000, Sigma-Aldrich).

    Article Title: Heterogeneous Nuclear Ribonucleoprotein (hnRNP) E1 Binds to hnRNP A2 and Inhibits Translation of A2 Response Element mRNAs
    Article Snippet: Cross-linking was initiated by exposure to a 302-nm 100-W UV lamp held 5 cm from the solutions for 5 min. Cross-linked samples were reduced by adding dithiothreitol to a final concentration of 100 mM and boiling for 5 min, which dissociates the biotin label from hnRNP A2 and leaves it attached to the cross-linked protein. .. Biotin-labeled proteins were separated by SDS-PAGE and either stained with Coomassie blue or blotted onto polyvinylidene difluoride (PVDF) membrane, probed with 20 ng/ml streptavidin-horseradish peroxidase for 90 min at room temperature, and developed by enhanced chemiluminescence (Pierce Chemical) to identify biotin-conjugated proteins.

    Article Title: F5-peptide induces aspermatogenesis by disrupting organization of actin- and microtubule-based cytoskeletons in the testis
    Article Snippet: Sections were blocked with 10% normal goat serum, incubated with α-tubulin or EB1 antibody ( ) overnight at 4°C, followed by an incubation with biotinylated secondary antibody, and then streptavidin-horseradish peroxidase (Life Technologies). .. Negative controls using the same concentration of normal mouse IgG to substitute the primary antibody or the omission of secondary antibody were also included in our experiments.

    Staining:

    Article Title: Carbohydrate Mimetic Peptides Augment Carbohydrate-Reactive Immune Responses in the Absence of Immune Pathology
    Article Snippet: Sections were then incubated with a biotinylated mixture of anti-mouse IgG and anti-mouse IgM (Dako Cytomation) for 1 hr at RT and washed in DPBS containing 0.1% Tween 20, followed by incubation with streptavidin horseradish peroxidase (1:5000, Pierce, Rockford, IL, USA) for 30 min at RT. .. To determine relative reactivity of IgG and IgM, slides were separately stained with biotinylated anti-murine IgG (Sigma, St. Louis, MO, USA, 1:400) and –IgM (Vector, Burlingame, CA, USA, 1:400) antibodies.

    Article Title: Heterogeneous Nuclear Ribonucleoprotein (hnRNP) E1 Binds to hnRNP A2 and Inhibits Translation of A2 Response Element mRNAs
    Article Snippet: .. Biotin-labeled proteins were separated by SDS-PAGE and either stained with Coomassie blue or blotted onto polyvinylidene difluoride (PVDF) membrane, probed with 20 ng/ml streptavidin-horseradish peroxidase for 90 min at room temperature, and developed by enhanced chemiluminescence (Pierce Chemical) to identify biotin-conjugated proteins. ..

    Article Title: Na+/H+ exchanger type 1 is a receptor for pathogenic subgroup J avian leukosis virus
    Article Snippet: The resulting Western blot was probed with streptavidin–horseradish peroxidase (Invitrogen). .. By using this approach, ≈8 × 107 unlabeled DF-1 cells were used to generate two distinct bands of ≈90 kDa that could be visualized by Sypro Ruby staining (Bio-Rad).

    Article Title: Functional test of PCDHB11, the most human-specific neuronal surface protein
    Article Snippet: For GFP detection, membranes were incubated with a rabbit polyclonal antibody (Life Technologies, A-11122) diluted 1:2000 in 50 mM Tris, 100 mM NaCl, 0.1 % Tween 20, pH 7.4, with 3 % bovine serum albumine (Sigma, St. Louis, MO) and a horse-radish peroxidase-conjugated anti-rabbit secondary antibody (Invitrogen, Waltham, MA), while for HA revelation, they were incubated with biotinylated 3 F10 rat monoclonal antibody (Roche Life Science, Indianapolis, IN) diluted 1:500 in the same buffer, and streptavidin-horseradish peroxidase (Invitrogen, Waltham, MA). .. For HA staining, live K562 cultures were incubated for one hour with the biotinylated 3 F10 anti-HA, then for 40 min with streptavidin-Alexa555, both diluted 1:250 in culture medium at 8 °C, then fixed for 15 min in 4 % paraformaldehyde, 4 % sucrose in phosphate-buffered saline at 8 °C, deposited on slides using a CytoSpin (Thermo Fisher Scientific) and mounted in ProLong containing 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen).

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  • 94
    Thermo Fisher horseradish peroxidase conjugated streptavidin
    Depletion of glypican-1 stimulates the endocytosis of PrP C . SH-SY5Y cells expressing wild type PrP C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP C . Cells were then lysed and total PrP C immunoprecipitated from the sample using antibody 3F4. ( A ) Samples were subjected to western blot analysis and the biotin-labelled PrP C fraction was detected with peroxidase-conjugated <t>streptavidin.</t> ( B ) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. ( C ) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP C in the cell lysates from (A). β-actin was used as a loading control. ( D ) SH-SY5Y cells expressing PrP C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( E ) Densitometric analysis of the proportion of total PrP C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. ( F ) SH-SY5Y cells expressing PrP C were seeded onto glass coverslips and grown to 50% confluency. Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P
    Horseradish Peroxidase Conjugated Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 90 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/horseradish peroxidase conjugated streptavidin/product/Thermo Fisher
    Average 94 stars, based on 90 article reviews
    Price from $9.99 to $1999.99
    horseradish peroxidase conjugated streptavidin - by Bioz Stars, 2020-04
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    99
    Thermo Fisher streptavidin hrp
    Schematic diagram showed non-radioactive metabolic incorporation followed by azide-biotin or azide-Alex555 labeling, and biotin signals of proteins were detected by <t>streptavidin-HRP</t> by western blot. HPG is incorporated into newly synthesized proteins by metabolism and protein synthesis and the triazole conjugation between newly alkyne proteins labeled HPG and azide labeled either biotin or Alex555 via CuSO4 catalysis (A) . (B-a) The detection of biotin signals from extracted total proteins labeled by labeling reaction. Normal culture medium was changed to replace DMEM free of L-methionine supplemented with HPG after pulse 4 hr, and proteins were extracted in each of group at various time points including 0, 4, 24 and 72 hr. (B-b) Biotin signals of total proteins were detected. 1: Normal culture condition group; 2: HPG plus anisomycin group; 3: HPG group. (B-c,d,e) Biotin signals of Bcl-2, MMP-9 and IgG were individually detected in the immunoprecipitate pulled down by primary antibodies via siRNA post-transfection followed by non-radioactive metabolic labeling. (B-f) Radioactive isotope 35 S-methonine incorporated into synthesized IgG purified by immunoprecipitation was detected by autoradiography. 1: 35 S-methonine treated human choriocarcinoma cell line BeWo group and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 2: cycloheximide plus 35 S-methonine treated BeWo group then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 3: 35 S-methonine treated skin fibroblast and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins.
    Streptavidin Hrp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 162 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin hrp/product/Thermo Fisher
    Average 99 stars, based on 162 article reviews
    Price from $9.99 to $1999.99
    streptavidin hrp - by Bioz Stars, 2020-04
    99/100 stars
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    Depletion of glypican-1 stimulates the endocytosis of PrP C . SH-SY5Y cells expressing wild type PrP C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP C . Cells were then lysed and total PrP C immunoprecipitated from the sample using antibody 3F4. ( A ) Samples were subjected to western blot analysis and the biotin-labelled PrP C fraction was detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. ( C ) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP C in the cell lysates from (A). β-actin was used as a loading control. ( D ) SH-SY5Y cells expressing PrP C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( E ) Densitometric analysis of the proportion of total PrP C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. ( F ) SH-SY5Y cells expressing PrP C were seeded onto glass coverslips and grown to 50% confluency. Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: Depletion of glypican-1 stimulates the endocytosis of PrP C . SH-SY5Y cells expressing wild type PrP C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP C . Cells were then lysed and total PrP C immunoprecipitated from the sample using antibody 3F4. ( A ) Samples were subjected to western blot analysis and the biotin-labelled PrP C fraction was detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. ( C ) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP C in the cell lysates from (A). β-actin was used as a loading control. ( D ) SH-SY5Y cells expressing PrP C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( E ) Densitometric analysis of the proportion of total PrP C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. ( F ) SH-SY5Y cells expressing PrP C were seeded onto glass coverslips and grown to 50% confluency. Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P

    Article Snippet: Where indicated, biotin-labelled PrP was detected by subsequent immunoprecipitation of epitope-tagged PrP from the individual fractions using antibody 3F4 (Eurogentec Ltd., Southampton, U.K.) and subsequent immunoblotting using horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Cramlington, U.K.).

    Techniques: Expressing, Incubation, Immunoprecipitation, Western Blot, Gradient Centrifugation, Microscopy

    Depletion of glypican-1 does not affect cell division or surface levels of PrP C . ( A ) ScN2a cells were seeded into 96 well plates and treated with transfection reagent only or incubated with either control siRNA or one of the four siRNAs targeted to glypican-1. Those experiments exceeding 48 h were dosed with a second treatment of the indicated siRNAs. Cells were then rinsed with PBS and fixed with 70% (v/v) ethanol. Plates were allowed to dry, stained with Hoescht 33342 and the fluorescence measured. ( B ) ScN2a cells were treated with control or glypican-1 siRNA. After 96 h, cell monolayers were labelled with a membrane impermeable biotin reagent. Biotin-labelled cell surface PrP was detected by immunoprecipitation using 6D11 and subsequent immunoblotting using HRP-conjugated streptavidin. Total PrP and PK-resistant PrP (PrP Sc ) were detected by immunoblotting using antibody 6D11. ( C ) Densitometric analysis of the proportion of the relative amount of biotinylated cell surface PrP in the absence or presence of glypican-1 siRNA from three independent experiments.

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: Depletion of glypican-1 does not affect cell division or surface levels of PrP C . ( A ) ScN2a cells were seeded into 96 well plates and treated with transfection reagent only or incubated with either control siRNA or one of the four siRNAs targeted to glypican-1. Those experiments exceeding 48 h were dosed with a second treatment of the indicated siRNAs. Cells were then rinsed with PBS and fixed with 70% (v/v) ethanol. Plates were allowed to dry, stained with Hoescht 33342 and the fluorescence measured. ( B ) ScN2a cells were treated with control or glypican-1 siRNA. After 96 h, cell monolayers were labelled with a membrane impermeable biotin reagent. Biotin-labelled cell surface PrP was detected by immunoprecipitation using 6D11 and subsequent immunoblotting using HRP-conjugated streptavidin. Total PrP and PK-resistant PrP (PrP Sc ) were detected by immunoblotting using antibody 6D11. ( C ) Densitometric analysis of the proportion of the relative amount of biotinylated cell surface PrP in the absence or presence of glypican-1 siRNA from three independent experiments.

    Article Snippet: Where indicated, biotin-labelled PrP was detected by subsequent immunoprecipitation of epitope-tagged PrP from the individual fractions using antibody 3F4 (Eurogentec Ltd., Southampton, U.K.) and subsequent immunoblotting using horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Cramlington, U.K.).

    Techniques: Transfection, Incubation, Staining, Fluorescence, Immunoprecipitation

    Heparin stimulates the endocytosis of PrP C in a dose-dependent manner and displaces it from detergent-resistant lipid rafts. ( A ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated for 1 h at 37°C in the absence or presence of various concentrations of heparin diluted in OptiMEM. Prior to lysis cells were, where indicated, incubated with trypsin to digest cell surface PrP C . Cells were then lysed and PrP C immunoprecipitated from the sample using antibody 3F4. Samples were subjected to SDS PAGE and western blot analysis and the biotin-labelled PrP C detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis of multiple blots from four separate experiments as described in (A) is shown. ( C ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP C was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to SDS-PAGE and western blotting. The gradient fractions from both the untreated and heparin treated cells were analysed on the same SDS gel and immunoblotted under identical conditions. The biotin-labelled PrP C was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( D ) Densitometric analysis of the proportion of total PrP C in the detergent soluble fractions of the plasma membrane. ( E ) Untransfected SH-SY5Y cells and SH-SY5Y cells expressing either PrP C or PrP-TM were grown to confluence and then incubated for 1 h in the presence or absence of 50 µM heparin prepared in OptiMEM. Media samples were collected and concentrated and cells harvested and lysed. Cell lysate samples were immunoblotted for PrP C using antibody 3F4, with β-actin used as a loading control. ( F ) Quantification of PrP C and PrP-TM levels after treatment of cells with heparin as in (E). Experiments were performed in triplicate and repeated on three occasions. * P

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: Heparin stimulates the endocytosis of PrP C in a dose-dependent manner and displaces it from detergent-resistant lipid rafts. ( A ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated for 1 h at 37°C in the absence or presence of various concentrations of heparin diluted in OptiMEM. Prior to lysis cells were, where indicated, incubated with trypsin to digest cell surface PrP C . Cells were then lysed and PrP C immunoprecipitated from the sample using antibody 3F4. Samples were subjected to SDS PAGE and western blot analysis and the biotin-labelled PrP C detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis of multiple blots from four separate experiments as described in (A) is shown. ( C ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP C was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to SDS-PAGE and western blotting. The gradient fractions from both the untreated and heparin treated cells were analysed on the same SDS gel and immunoblotted under identical conditions. The biotin-labelled PrP C was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( D ) Densitometric analysis of the proportion of total PrP C in the detergent soluble fractions of the plasma membrane. ( E ) Untransfected SH-SY5Y cells and SH-SY5Y cells expressing either PrP C or PrP-TM were grown to confluence and then incubated for 1 h in the presence or absence of 50 µM heparin prepared in OptiMEM. Media samples were collected and concentrated and cells harvested and lysed. Cell lysate samples were immunoblotted for PrP C using antibody 3F4, with β-actin used as a loading control. ( F ) Quantification of PrP C and PrP-TM levels after treatment of cells with heparin as in (E). Experiments were performed in triplicate and repeated on three occasions. * P

    Article Snippet: Where indicated, biotin-labelled PrP was detected by subsequent immunoprecipitation of epitope-tagged PrP from the individual fractions using antibody 3F4 (Eurogentec Ltd., Southampton, U.K.) and subsequent immunoblotting using horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Cramlington, U.K.).

    Techniques: Expressing, Incubation, Lysis, Immunoprecipitation, SDS Page, Western Blot, Gradient Centrifugation, SDS-Gel

    Depletion of glypican-1 inhibits the association of PrP-TM with DRMs. SH-SY5Y cells expressing PrP-TM were treated with either control siRNA or siRNA targeted to glypican-1 and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C in the presence of Tyrphostin A23 to block endocytosis. The media was removed and the cells washed in phosphate-buffered saline prior to homogenisation in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( A ) Quantification of glypican-1 and PrP-TM expression in cell lysates. To detect glypican-1, cell lysate samples were treated with heparinase I and heparinase III prior to electrophoresis as described in the materials and methods section. ( B ) PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and then subjected to western blotting with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after siRNA treatment from multiple blots from three independent experiments. * P

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: Depletion of glypican-1 inhibits the association of PrP-TM with DRMs. SH-SY5Y cells expressing PrP-TM were treated with either control siRNA or siRNA targeted to glypican-1 and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C in the presence of Tyrphostin A23 to block endocytosis. The media was removed and the cells washed in phosphate-buffered saline prior to homogenisation in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( A ) Quantification of glypican-1 and PrP-TM expression in cell lysates. To detect glypican-1, cell lysate samples were treated with heparinase I and heparinase III prior to electrophoresis as described in the materials and methods section. ( B ) PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and then subjected to western blotting with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after siRNA treatment from multiple blots from three independent experiments. * P

    Article Snippet: Where indicated, biotin-labelled PrP was detected by subsequent immunoprecipitation of epitope-tagged PrP from the individual fractions using antibody 3F4 (Eurogentec Ltd., Southampton, U.K.) and subsequent immunoblotting using horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Cramlington, U.K.).

    Techniques: Expressing, Incubation, Blocking Assay, Homogenization, Gradient Centrifugation, Electrophoresis, Immunoprecipitation, Western Blot

    The association of PrP-TM with DRMs is disrupted by treatment of cells with either heparin or bacterial PI-PLC. SH-SY5Y cells expressing PrP-TM were surface biotinylated and then ( A ) incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C or ( B ) incubated in the absence or presence of 1 U/ml bacterial PI-PLC for 1 h at 4°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to western blotting. The biotin-labelled PrP-TM fraction was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after heparin and PI-PLC treatment. Experiments were performed in triplicate and repeated on three occasions. * P

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: The association of PrP-TM with DRMs is disrupted by treatment of cells with either heparin or bacterial PI-PLC. SH-SY5Y cells expressing PrP-TM were surface biotinylated and then ( A ) incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C or ( B ) incubated in the absence or presence of 1 U/ml bacterial PI-PLC for 1 h at 4°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to western blotting. The biotin-labelled PrP-TM fraction was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after heparin and PI-PLC treatment. Experiments were performed in triplicate and repeated on three occasions. * P

    Article Snippet: Where indicated, biotin-labelled PrP was detected by subsequent immunoprecipitation of epitope-tagged PrP from the individual fractions using antibody 3F4 (Eurogentec Ltd., Southampton, U.K.) and subsequent immunoblotting using horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Cramlington, U.K.).

    Techniques: Planar Chromatography, Expressing, Incubation, Gradient Centrifugation, Immunoprecipitation, Western Blot

    Schematic diagram showed non-radioactive metabolic incorporation followed by azide-biotin or azide-Alex555 labeling, and biotin signals of proteins were detected by streptavidin-HRP by western blot. HPG is incorporated into newly synthesized proteins by metabolism and protein synthesis and the triazole conjugation between newly alkyne proteins labeled HPG and azide labeled either biotin or Alex555 via CuSO4 catalysis (A) . (B-a) The detection of biotin signals from extracted total proteins labeled by labeling reaction. Normal culture medium was changed to replace DMEM free of L-methionine supplemented with HPG after pulse 4 hr, and proteins were extracted in each of group at various time points including 0, 4, 24 and 72 hr. (B-b) Biotin signals of total proteins were detected. 1: Normal culture condition group; 2: HPG plus anisomycin group; 3: HPG group. (B-c,d,e) Biotin signals of Bcl-2, MMP-9 and IgG were individually detected in the immunoprecipitate pulled down by primary antibodies via siRNA post-transfection followed by non-radioactive metabolic labeling. (B-f) Radioactive isotope 35 S-methonine incorporated into synthesized IgG purified by immunoprecipitation was detected by autoradiography. 1: 35 S-methonine treated human choriocarcinoma cell line BeWo group and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 2: cycloheximide plus 35 S-methonine treated BeWo group then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 3: 35 S-methonine treated skin fibroblast and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins.

    Journal: BMC Cell Biology

    Article Title: A morphologic and semi-quantitative technique to analyze synthesis and release of specific proteins in cells

    doi: 10.1186/s12860-014-0045-1

    Figure Lengend Snippet: Schematic diagram showed non-radioactive metabolic incorporation followed by azide-biotin or azide-Alex555 labeling, and biotin signals of proteins were detected by streptavidin-HRP by western blot. HPG is incorporated into newly synthesized proteins by metabolism and protein synthesis and the triazole conjugation between newly alkyne proteins labeled HPG and azide labeled either biotin or Alex555 via CuSO4 catalysis (A) . (B-a) The detection of biotin signals from extracted total proteins labeled by labeling reaction. Normal culture medium was changed to replace DMEM free of L-methionine supplemented with HPG after pulse 4 hr, and proteins were extracted in each of group at various time points including 0, 4, 24 and 72 hr. (B-b) Biotin signals of total proteins were detected. 1: Normal culture condition group; 2: HPG plus anisomycin group; 3: HPG group. (B-c,d,e) Biotin signals of Bcl-2, MMP-9 and IgG were individually detected in the immunoprecipitate pulled down by primary antibodies via siRNA post-transfection followed by non-radioactive metabolic labeling. (B-f) Radioactive isotope 35 S-methonine incorporated into synthesized IgG purified by immunoprecipitation was detected by autoradiography. 1: 35 S-methonine treated human choriocarcinoma cell line BeWo group and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 2: cycloheximide plus 35 S-methonine treated BeWo group then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 3: 35 S-methonine treated skin fibroblast and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins.

    Article Snippet: Each diluted standard was incubated with primary antibody coated on the well and streptavidin-HRP and OD value for each sample was detected by ELISA.

    Techniques: Labeling, Western Blot, Synthesized, Conjugation Assay, Transfection, Purification, Immunoprecipitation, Autoradiography

    Electron microscopy of PA1b-bound V-ATPase. A , representative classes of PA1b-streptavidin-HRP-bound V-ATPase in the absence of ATP. B , as A but in the presence of 2 m m Mg·ATP. The PA1b-streptavidin-HRP density is indicated by an arrow in the far left panel 1 of A. Scale bars in both A and B represent 15 nm. C–E , three-dimensional reconstructions of the V-ATPase viewed perpendicular to the long axis of the complex ( upper image ) and from the extracellular end ( lower image ) bound to PA1b ( C ), bound to PA1b after the addition of Mg·ATP ( D ) and a control with no PA1b ( E ). All models were generated using EMAN, and the picture was produced using Chimera rendered at the same sigma level. In C ( lower ), the decameric c ring (Protein Data Bank ID code 2DB4 ( 53 ) r ainbow colors ) and a subunit model ( red ) have been fitted to the PA1b-streptavidin-HRP V-ATPase reconstruction in the absence of ATP using Chimera. If catalytically active, the c ring would rotate counterclockwise with respect to subunit a when observed from this perspective.

    Journal: The Journal of Biological Chemistry

    Article Title: PA1b Inhibitor Binding to Subunits c and e of the Vacuolar ATPase Reveals Its Insecticidal Mechanism *

    doi: 10.1074/jbc.M113.541250

    Figure Lengend Snippet: Electron microscopy of PA1b-bound V-ATPase. A , representative classes of PA1b-streptavidin-HRP-bound V-ATPase in the absence of ATP. B , as A but in the presence of 2 m m Mg·ATP. The PA1b-streptavidin-HRP density is indicated by an arrow in the far left panel 1 of A. Scale bars in both A and B represent 15 nm. C–E , three-dimensional reconstructions of the V-ATPase viewed perpendicular to the long axis of the complex ( upper image ) and from the extracellular end ( lower image ) bound to PA1b ( C ), bound to PA1b after the addition of Mg·ATP ( D ) and a control with no PA1b ( E ). All models were generated using EMAN, and the picture was produced using Chimera rendered at the same sigma level. In C ( lower ), the decameric c ring (Protein Data Bank ID code 2DB4 ( 53 ) r ainbow colors ) and a subunit model ( red ) have been fitted to the PA1b-streptavidin-HRP V-ATPase reconstruction in the absence of ATP using Chimera. If catalytically active, the c ring would rotate counterclockwise with respect to subunit a when observed from this perspective.

    Article Snippet: For the second experiment, 4 μl of V-ATPase (4 μg) was mixed with 3 μl of biotin-PA1b (3 μg) and 3 μl of streptavidin-HRP (15 μg), made up to 60 μl using V-ATPase buffer and incubated for 30 min. Mg·ATP was from a stock solution of 100 mm at pH 7.5 to a final concentration of 5 mm , and the mixture was incubated at room temperature for 5 min to allow for complete turnover.

    Techniques: Electron Microscopy, Generated, Produced

    Expression of recombinant porcine CCL2. (A) CHO cell line stably expressing the porcine CCL2 fused to GFP. The expression of GFP fusion protein was directly analysed by flow cytometry. Non transfected CHO cells were used as negative control (grey histogram). 5 000 cells were acquired. (B) Western blot of CCL2-GFP produced by transfected CHO cells. Different dilutions of supernatant were resolved by 15% SDS-PAGE under reducing conditions and revealed with biotinylated anti-GFP and streptavidin-HRP. Numbers on the left indicate the position of MW markers. (C) Chemotactic activity of CCL2-GFP on porcine blood monocytes. Chemotaxis was assessed with the Transwell cell migration system and subsequent flow cytometry counting of migrated cells by a 45 s acquisition. (1) FSC versus SSC dot plot of migrated cells in response to supernatants from CHO cells expressing CCL2-GFP or the inverted sequence of pCCL2 fused to GFP (InvCCL2-GFP, negative control). (2) Results expressed as migration index, calculated as the ratio of the number of cells migrating to the chemokine and the number of cells in the negative control. Results from one representative experiment out of three performed are shown. (A color version of this figure is available at www.vetres.org. )

    Journal: Veterinary Research

    Article Title: Porcine monocyte subsets differ in the expression of CCR2 and in their responsiveness to CCL2

    doi: 10.1051/vetres/2010048

    Figure Lengend Snippet: Expression of recombinant porcine CCL2. (A) CHO cell line stably expressing the porcine CCL2 fused to GFP. The expression of GFP fusion protein was directly analysed by flow cytometry. Non transfected CHO cells were used as negative control (grey histogram). 5 000 cells were acquired. (B) Western blot of CCL2-GFP produced by transfected CHO cells. Different dilutions of supernatant were resolved by 15% SDS-PAGE under reducing conditions and revealed with biotinylated anti-GFP and streptavidin-HRP. Numbers on the left indicate the position of MW markers. (C) Chemotactic activity of CCL2-GFP on porcine blood monocytes. Chemotaxis was assessed with the Transwell cell migration system and subsequent flow cytometry counting of migrated cells by a 45 s acquisition. (1) FSC versus SSC dot plot of migrated cells in response to supernatants from CHO cells expressing CCL2-GFP or the inverted sequence of pCCL2 fused to GFP (InvCCL2-GFP, negative control). (2) Results expressed as migration index, calculated as the ratio of the number of cells migrating to the chemokine and the number of cells in the negative control. Results from one representative experiment out of three performed are shown. (A color version of this figure is available at www.vetres.org. )

    Article Snippet: The expression of GFP-fused proteins in these clones was confirmed by Western blot using a biotin-conjugated goat anti-GFP polyclonal antibody and streptavidin-HRP.

    Techniques: Expressing, Recombinant, Stable Transfection, Flow Cytometry, Cytometry, Transfection, Negative Control, Western Blot, Produced, SDS Page, Activity Assay, Chemotaxis Assay, Migration, Sequencing