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PKA and PLB binding to AKAP7 γ do not interfere with AKAP7 γ dimerization. (a) Lysates from either AKAP7 γ -EGFP or AKAP7 γ -ΔPKA-EGFP transfected HEK-293 cells were subjected to pulldown assays using bacterially purified S-tagged AKAP7 γ proteins precharged on S-protein resin; full length AKAP7 γ or AKAP7 γ -1–268 (which lacks the PKA binding domain). Anti-GFP antibody was used for detecting protein interactions by western blot analysis (upper panel). Protein stain of the nitrocellulose membrane before western blot analysis is shown in the lower panel. Input from the transfected cells (20 μ L) is shown in the middle panel; n = 3. (b) Lysates from AKAP7 γ -mCherry transfected HEK-293 cells were subjected to pulldown assays using bacterially purified S-tagged AKAP7 γ precharged on S-protein resin. After an overnight incubation, the pulldowns were washed extensively and then incubated with the PKA anchoring disrupting peptide AKAPIS or control, scrambled peptide (10 μ M) for 3 hours. The pulldowns were washed again, and association of AKAP7 γ was determined by anti-mCherry antibody (upper panel). Protein stain of the nitrocellulose membrane before western blot analysis is shown in the lower panel. Input from the transfected cells (20 μ L) is shown in the middle panel; n = 3. (c) The AKAP18 γ peptide array membranes used in (c) were stripped for 45 min in 62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 100 mM β -mercaptoethanol at 60°C and washed three times in <t>TBST</t> for 10 min before being blocked in 1% casein (in TBST) overnight at 4°C. Concomitantly, 2 µ g/mL of recombinant human His-S-AKAP18 γ protein was preincubated with or without 10 µ M phospholamban peptide (1–30) overnight at 4°C, before being overlaid onto the peptide array membranes for 2 hours at room temperature. The membranes were washed, incubated with <t>HRP-conjugated</t> anti-S-tag antibody, and developed as described above.
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1) Product Images from "Analysis of AKAP7 γ Dimerization"

Article Title: Analysis of AKAP7 γ Dimerization

Journal: Journal of Signal Transduction

doi: 10.1155/2015/371626

PKA and PLB binding to AKAP7 γ do not interfere with AKAP7 γ dimerization. (a) Lysates from either AKAP7 γ -EGFP or AKAP7 γ -ΔPKA-EGFP transfected HEK-293 cells were subjected to pulldown assays using bacterially purified S-tagged AKAP7 γ proteins precharged on S-protein resin; full length AKAP7 γ or AKAP7 γ -1–268 (which lacks the PKA binding domain). Anti-GFP antibody was used for detecting protein interactions by western blot analysis (upper panel). Protein stain of the nitrocellulose membrane before western blot analysis is shown in the lower panel. Input from the transfected cells (20 μ L) is shown in the middle panel; n = 3. (b) Lysates from AKAP7 γ -mCherry transfected HEK-293 cells were subjected to pulldown assays using bacterially purified S-tagged AKAP7 γ precharged on S-protein resin. After an overnight incubation, the pulldowns were washed extensively and then incubated with the PKA anchoring disrupting peptide AKAPIS or control, scrambled peptide (10 μ M) for 3 hours. The pulldowns were washed again, and association of AKAP7 γ was determined by anti-mCherry antibody (upper panel). Protein stain of the nitrocellulose membrane before western blot analysis is shown in the lower panel. Input from the transfected cells (20 μ L) is shown in the middle panel; n = 3. (c) The AKAP18 γ peptide array membranes used in (c) were stripped for 45 min in 62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 100 mM β -mercaptoethanol at 60°C and washed three times in TBST for 10 min before being blocked in 1% casein (in TBST) overnight at 4°C. Concomitantly, 2 µ g/mL of recombinant human His-S-AKAP18 γ protein was preincubated with or without 10 µ M phospholamban peptide (1–30) overnight at 4°C, before being overlaid onto the peptide array membranes for 2 hours at room temperature. The membranes were washed, incubated with HRP-conjugated anti-S-tag antibody, and developed as described above.
Figure Legend Snippet: PKA and PLB binding to AKAP7 γ do not interfere with AKAP7 γ dimerization. (a) Lysates from either AKAP7 γ -EGFP or AKAP7 γ -ΔPKA-EGFP transfected HEK-293 cells were subjected to pulldown assays using bacterially purified S-tagged AKAP7 γ proteins precharged on S-protein resin; full length AKAP7 γ or AKAP7 γ -1–268 (which lacks the PKA binding domain). Anti-GFP antibody was used for detecting protein interactions by western blot analysis (upper panel). Protein stain of the nitrocellulose membrane before western blot analysis is shown in the lower panel. Input from the transfected cells (20 μ L) is shown in the middle panel; n = 3. (b) Lysates from AKAP7 γ -mCherry transfected HEK-293 cells were subjected to pulldown assays using bacterially purified S-tagged AKAP7 γ precharged on S-protein resin. After an overnight incubation, the pulldowns were washed extensively and then incubated with the PKA anchoring disrupting peptide AKAPIS or control, scrambled peptide (10 μ M) for 3 hours. The pulldowns were washed again, and association of AKAP7 γ was determined by anti-mCherry antibody (upper panel). Protein stain of the nitrocellulose membrane before western blot analysis is shown in the lower panel. Input from the transfected cells (20 μ L) is shown in the middle panel; n = 3. (c) The AKAP18 γ peptide array membranes used in (c) were stripped for 45 min in 62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 100 mM β -mercaptoethanol at 60°C and washed three times in TBST for 10 min before being blocked in 1% casein (in TBST) overnight at 4°C. Concomitantly, 2 µ g/mL of recombinant human His-S-AKAP18 γ protein was preincubated with or without 10 µ M phospholamban peptide (1–30) overnight at 4°C, before being overlaid onto the peptide array membranes for 2 hours at room temperature. The membranes were washed, incubated with HRP-conjugated anti-S-tag antibody, and developed as described above.

Techniques Used: Binding Assay, Transfection, Purification, Western Blot, Staining, Incubation, Peptide Microarray, Recombinant

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Imaging:

Article Title: Reduced glucocerebrosidase activity in monocytes from patients with Parkinson’s disease
Article Snippet: .. Membranes were then washed in TBST (5 × 10 min) and incubated with anti-rabbit horseradish peroxidase (HRP) secondary antibody (Abcam) at 1:5000 dilution in 2.5% skim milk in TBST or anti-mouse Alexa Fluor 647 fluorescent secondary antibody (Abcam) in TBST for 2 h. A Chemidoc MP Imaging system (Biorad) was used for detection and immunoblot quantitation was performed using Imagelab software 5.2.1 (Biorad). ..

Incubation:

Article Title: Reduced glucocerebrosidase activity in monocytes from patients with Parkinson’s disease
Article Snippet: .. Membranes were then washed in TBST (5 × 10 min) and incubated with anti-rabbit horseradish peroxidase (HRP) secondary antibody (Abcam) at 1:5000 dilution in 2.5% skim milk in TBST or anti-mouse Alexa Fluor 647 fluorescent secondary antibody (Abcam) in TBST for 2 h. A Chemidoc MP Imaging system (Biorad) was used for detection and immunoblot quantitation was performed using Imagelab software 5.2.1 (Biorad). ..

Article Title: The anti-inflammatory effect of Sonchus oleraceus aqueous extract on lipopolysaccharide stimulated RAW 264.7 cells and mice
Article Snippet: .. Under gentle shaking, the blots were first blocked in 5% nonfat milk for 1 h. After washing in TBST three times, the blots were incubated with NF-κB primary antibody (Abcam, MA, 1:5000 dilution) and anti-β actin antibody (TransGen Biotech, Beijing, China, 1:5000 dilution) overnight at 4 °C. .. After washing in TBST three times, the blots were incubated with the subsequently secondary antibodies (Goat anti-rabbit IgG, HRP conjugated, Zhuangzhibio, Xi’an, China, 1:2000 dilution) at 4 °C for 50 min under gentle shaking.

Article Title: Knockdown of CD44 inhibits the invasion and metastasis of hepatocellular carcinoma both in vitro and in vivo by reversing epithelial-mesenchymal transition
Article Snippet: .. After being washed with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Abcam) for 1 h at room temperature. .. Proteins were detected in the ChemiDocTM XRS+ using the Image LabTM software (Bio-Rad, Hercules, CA, USA) after three washes with TBST.

Article Title: Estradiol promotes the progression of ER+ breast cancer through methylation-mediated RSK4 inactivation
Article Snippet: .. Next, the membranes were immersed into 5% non-fat milk diluted in TBST for 1 h at room temperature, followed by incubation with the indicated primary antibodies overnight at 4 °C, including anti‑RSK4 (1:2000 dilution; No.ab76117, Abcam), anti‑SP1 (1:1000 dilution; No. ab227383, Abcam), anti-DNMT1 (1:2000 dilution; No. #5032, Cell Signaling Technology, MA, USA), anti-DNMT3A (1:2000 dilution; No. #2160, Cell Signaling Technology), anti-DNMT3B (1:2000 dilution; No. #67,259, Cell Signaling Technology), anti-Ub (1:1000 dilution; No. #3933, Cell Signaling Technology) and anti-GAPDH (No. ab181602, Abcam). .. After being washed with Tris Buffered Saline with Tween-20 (TBST) for 3 times, the membranes were probed with the corresponding secondary antibodies at room temperature for 1 h. Band intensity was measured using chemiluminescent reagents (Millipore) and quantified by ImageJ software (verson1.48, National Institutes of Health, MD, USA).

Article Title: Epac1 inhibits PKR to reduce NLRP3 inflammasome proteins in retinal endothelial cells
Article Snippet: .. After blocking in TBST (10 mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 0.1% Tween 20) and 5% (w/v) BSA, membranes are treated with Epac1, phosphorylated PKR, total PKR, NLRP3, cleaved caspase 1, and IL-1β (Abcam, Cambridge, MA) or beta-actin (Santa Cruz Biotechnology, Santa Cruz, CA) primary antibodies followed by incubation with secondary antibodies labeled with horseradish peroxidase. .. A chemiluminescence reagent kit (Thermo Scientific, Pittsburgh, PA) was used to visualize antigen–antibody complexes.

Article Title: Cross-Regulation between TDP-43 and Paraspeckles Promotes Pluripotency-Differentiation Transition
Article Snippet: .. The coverslips were subsequently washed with TBST (1 × TBS containing 0.1% Tween 20) and incubated with 1 × blocking solution (Blocking reagent [Roche] diluted with TBST) for blocking at RT for 1 h. Then, the coverslips were incubated with primary antibodies, Anti-Digoxigenin mouse monoclonal antibody (clone 21H8, Abcam), Anti-TDP-43 rabbit polyclonal antibody (Proteintech, 1:100), anti-PSPC1 rabbit polyclonal antibody (1:1000; ) in 1 × blocking solution at room temperature for 1 h, washed three times with TBST for 5 min, and incubated with secondary antibodies, anti-mouse IgG Alexa 488, and anti-rabbit IgG, Alexa-568 (both Thermo Fisher Scientific]) in 1 × blocking solution at RT for 30 min, and washed three times with TBST for 5 min. .. The coverslips were mounted with VECTASHIELD Hard Set Mounting Medium with DAPI (Vector).

Blocking Assay:

Article Title: Epac1 inhibits PKR to reduce NLRP3 inflammasome proteins in retinal endothelial cells
Article Snippet: .. After blocking in TBST (10 mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 0.1% Tween 20) and 5% (w/v) BSA, membranes are treated with Epac1, phosphorylated PKR, total PKR, NLRP3, cleaved caspase 1, and IL-1β (Abcam, Cambridge, MA) or beta-actin (Santa Cruz Biotechnology, Santa Cruz, CA) primary antibodies followed by incubation with secondary antibodies labeled with horseradish peroxidase. .. A chemiluminescence reagent kit (Thermo Scientific, Pittsburgh, PA) was used to visualize antigen–antibody complexes.

Article Title: Cross-Regulation between TDP-43 and Paraspeckles Promotes Pluripotency-Differentiation Transition
Article Snippet: .. The coverslips were subsequently washed with TBST (1 × TBS containing 0.1% Tween 20) and incubated with 1 × blocking solution (Blocking reagent [Roche] diluted with TBST) for blocking at RT for 1 h. Then, the coverslips were incubated with primary antibodies, Anti-Digoxigenin mouse monoclonal antibody (clone 21H8, Abcam), Anti-TDP-43 rabbit polyclonal antibody (Proteintech, 1:100), anti-PSPC1 rabbit polyclonal antibody (1:1000; ) in 1 × blocking solution at room temperature for 1 h, washed three times with TBST for 5 min, and incubated with secondary antibodies, anti-mouse IgG Alexa 488, and anti-rabbit IgG, Alexa-568 (both Thermo Fisher Scientific]) in 1 × blocking solution at RT for 30 min, and washed three times with TBST for 5 min. .. The coverslips were mounted with VECTASHIELD Hard Set Mounting Medium with DAPI (Vector).

Quantitation Assay:

Article Title: Reduced glucocerebrosidase activity in monocytes from patients with Parkinson’s disease
Article Snippet: .. Membranes were then washed in TBST (5 × 10 min) and incubated with anti-rabbit horseradish peroxidase (HRP) secondary antibody (Abcam) at 1:5000 dilution in 2.5% skim milk in TBST or anti-mouse Alexa Fluor 647 fluorescent secondary antibody (Abcam) in TBST for 2 h. A Chemidoc MP Imaging system (Biorad) was used for detection and immunoblot quantitation was performed using Imagelab software 5.2.1 (Biorad). ..

Labeling:

Article Title: Epac1 inhibits PKR to reduce NLRP3 inflammasome proteins in retinal endothelial cells
Article Snippet: .. After blocking in TBST (10 mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 0.1% Tween 20) and 5% (w/v) BSA, membranes are treated with Epac1, phosphorylated PKR, total PKR, NLRP3, cleaved caspase 1, and IL-1β (Abcam, Cambridge, MA) or beta-actin (Santa Cruz Biotechnology, Santa Cruz, CA) primary antibodies followed by incubation with secondary antibodies labeled with horseradish peroxidase. .. A chemiluminescence reagent kit (Thermo Scientific, Pittsburgh, PA) was used to visualize antigen–antibody complexes.

Software:

Article Title: Reduced glucocerebrosidase activity in monocytes from patients with Parkinson’s disease
Article Snippet: .. Membranes were then washed in TBST (5 × 10 min) and incubated with anti-rabbit horseradish peroxidase (HRP) secondary antibody (Abcam) at 1:5000 dilution in 2.5% skim milk in TBST or anti-mouse Alexa Fluor 647 fluorescent secondary antibody (Abcam) in TBST for 2 h. A Chemidoc MP Imaging system (Biorad) was used for detection and immunoblot quantitation was performed using Imagelab software 5.2.1 (Biorad). ..

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    PKA and PLB binding to AKAP7 γ do not interfere with AKAP7 γ dimerization. (a) Lysates from either AKAP7 γ -EGFP or AKAP7 γ -ΔPKA-EGFP transfected HEK-293 cells were subjected to pulldown assays using bacterially purified S-tagged AKAP7 γ proteins precharged on S-protein resin; full length AKAP7 γ or AKAP7 γ -1–268 (which lacks the PKA binding domain). Anti-GFP antibody was used for detecting protein interactions by western blot analysis (upper panel). Protein stain of the nitrocellulose membrane before western blot analysis is shown in the lower panel. Input from the transfected cells (20 μ L) is shown in the middle panel; n = 3. (b) Lysates from AKAP7 γ -mCherry transfected HEK-293 cells were subjected to pulldown assays using bacterially purified S-tagged AKAP7 γ precharged on S-protein resin. After an overnight incubation, the pulldowns were washed extensively and then incubated with the PKA anchoring disrupting peptide AKAPIS or control, scrambled peptide (10 μ M) for 3 hours. The pulldowns were washed again, and association of AKAP7 γ was determined by anti-mCherry antibody (upper panel). Protein stain of the nitrocellulose membrane before western blot analysis is shown in the lower panel. Input from the transfected cells (20 μ L) is shown in the middle panel; n = 3. (c) The AKAP18 γ peptide array membranes used in (c) were stripped for 45 min in 62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 100 mM β -mercaptoethanol at 60°C and washed three times in <t>TBST</t> for 10 min before being blocked in 1% casein (in TBST) overnight at 4°C. Concomitantly, 2 µ g/mL of recombinant human His-S-AKAP18 γ protein was preincubated with or without 10 µ M phospholamban peptide (1–30) overnight at 4°C, before being overlaid onto the peptide array membranes for 2 hours at room temperature. The membranes were washed, incubated with <t>HRP-conjugated</t> anti-S-tag antibody, and developed as described above.
    Tbst, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 1125 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tbst/product/Abcam
    Average 92 stars, based on 1125 article reviews
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    Abcam tris buffered saline tween tbst
    PKA and PLB binding to AKAP7 γ do not interfere with AKAP7 γ dimerization. (a) Lysates from either AKAP7 γ -EGFP or AKAP7 γ -ΔPKA-EGFP transfected HEK-293 cells were subjected to pulldown assays using bacterially purified S-tagged AKAP7 γ proteins precharged on S-protein resin; full length AKAP7 γ or AKAP7 γ -1–268 (which lacks the PKA binding domain). Anti-GFP antibody was used for detecting protein interactions by western blot analysis (upper panel). Protein stain of the nitrocellulose membrane before western blot analysis is shown in the lower panel. Input from the transfected cells (20 μ L) is shown in the middle panel; n = 3. (b) Lysates from AKAP7 γ -mCherry transfected HEK-293 cells were subjected to pulldown assays using bacterially purified S-tagged AKAP7 γ precharged on S-protein resin. After an overnight incubation, the pulldowns were washed extensively and then incubated with the PKA anchoring disrupting peptide AKAPIS or control, scrambled peptide (10 μ M) for 3 hours. The pulldowns were washed again, and association of AKAP7 γ was determined by anti-mCherry antibody (upper panel). Protein stain of the nitrocellulose membrane before western blot analysis is shown in the lower panel. Input from the transfected cells (20 μ L) is shown in the middle panel; n = 3. (c) The AKAP18 γ peptide array membranes used in (c) were stripped for 45 min in 62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 100 mM β -mercaptoethanol at 60°C and washed three times in <t>TBST</t> for 10 min before being blocked in 1% casein (in TBST) overnight at 4°C. Concomitantly, 2 µ g/mL of recombinant human His-S-AKAP18 γ protein was preincubated with or without 10 µ M phospholamban peptide (1–30) overnight at 4°C, before being overlaid onto the peptide array membranes for 2 hours at room temperature. The membranes were washed, incubated with <t>HRP-conjugated</t> anti-S-tag antibody, and developed as described above.
    Tris Buffered Saline Tween Tbst, supplied by Abcam, used in various techniques. Bioz Stars score: 91/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PKA and PLB binding to AKAP7 γ do not interfere with AKAP7 γ dimerization. (a) Lysates from either AKAP7 γ -EGFP or AKAP7 γ -ΔPKA-EGFP transfected HEK-293 cells were subjected to pulldown assays using bacterially purified S-tagged AKAP7 γ proteins precharged on S-protein resin; full length AKAP7 γ or AKAP7 γ -1–268 (which lacks the PKA binding domain). Anti-GFP antibody was used for detecting protein interactions by western blot analysis (upper panel). Protein stain of the nitrocellulose membrane before western blot analysis is shown in the lower panel. Input from the transfected cells (20 μ L) is shown in the middle panel; n = 3. (b) Lysates from AKAP7 γ -mCherry transfected HEK-293 cells were subjected to pulldown assays using bacterially purified S-tagged AKAP7 γ precharged on S-protein resin. After an overnight incubation, the pulldowns were washed extensively and then incubated with the PKA anchoring disrupting peptide AKAPIS or control, scrambled peptide (10 μ M) for 3 hours. The pulldowns were washed again, and association of AKAP7 γ was determined by anti-mCherry antibody (upper panel). Protein stain of the nitrocellulose membrane before western blot analysis is shown in the lower panel. Input from the transfected cells (20 μ L) is shown in the middle panel; n = 3. (c) The AKAP18 γ peptide array membranes used in (c) were stripped for 45 min in 62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 100 mM β -mercaptoethanol at 60°C and washed three times in TBST for 10 min before being blocked in 1% casein (in TBST) overnight at 4°C. Concomitantly, 2 µ g/mL of recombinant human His-S-AKAP18 γ protein was preincubated with or without 10 µ M phospholamban peptide (1–30) overnight at 4°C, before being overlaid onto the peptide array membranes for 2 hours at room temperature. The membranes were washed, incubated with HRP-conjugated anti-S-tag antibody, and developed as described above.

    Journal: Journal of Signal Transduction

    Article Title: Analysis of AKAP7 γ Dimerization

    doi: 10.1155/2015/371626

    Figure Lengend Snippet: PKA and PLB binding to AKAP7 γ do not interfere with AKAP7 γ dimerization. (a) Lysates from either AKAP7 γ -EGFP or AKAP7 γ -ΔPKA-EGFP transfected HEK-293 cells were subjected to pulldown assays using bacterially purified S-tagged AKAP7 γ proteins precharged on S-protein resin; full length AKAP7 γ or AKAP7 γ -1–268 (which lacks the PKA binding domain). Anti-GFP antibody was used for detecting protein interactions by western blot analysis (upper panel). Protein stain of the nitrocellulose membrane before western blot analysis is shown in the lower panel. Input from the transfected cells (20 μ L) is shown in the middle panel; n = 3. (b) Lysates from AKAP7 γ -mCherry transfected HEK-293 cells were subjected to pulldown assays using bacterially purified S-tagged AKAP7 γ precharged on S-protein resin. After an overnight incubation, the pulldowns were washed extensively and then incubated with the PKA anchoring disrupting peptide AKAPIS or control, scrambled peptide (10 μ M) for 3 hours. The pulldowns were washed again, and association of AKAP7 γ was determined by anti-mCherry antibody (upper panel). Protein stain of the nitrocellulose membrane before western blot analysis is shown in the lower panel. Input from the transfected cells (20 μ L) is shown in the middle panel; n = 3. (c) The AKAP18 γ peptide array membranes used in (c) were stripped for 45 min in 62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 100 mM β -mercaptoethanol at 60°C and washed three times in TBST for 10 min before being blocked in 1% casein (in TBST) overnight at 4°C. Concomitantly, 2 µ g/mL of recombinant human His-S-AKAP18 γ protein was preincubated with or without 10 µ M phospholamban peptide (1–30) overnight at 4°C, before being overlaid onto the peptide array membranes for 2 hours at room temperature. The membranes were washed, incubated with HRP-conjugated anti-S-tag antibody, and developed as described above.

    Article Snippet: Thereafter the membranes were washed five times in TBST for 5 min before incubation with HRP-conjugated anti-S-tag antibody (Abcam, ab18589, 1 : 2500) for 1 hour at room temperature.

    Techniques: Binding Assay, Transfection, Purification, Western Blot, Staining, Incubation, Peptide Microarray, Recombinant