WB analysis and immunoprecipitation were conducted as previously depicted [30 (link)]. If necessary, membranes were stripped using stripping buffer (Bio-Rad, Hercules, CA, USA) and reprobed with appropriate antibodies.
Stripping buffer
Manufactured by Bio-Rad
Sourced in United States
Stripping buffer is a laboratory solution used to remove primary antibodies from western blot membranes. It facilitates the reuse of the membrane for subsequent probing with different antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin antibody, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Sirt1, by Cell Signaling Technology (1 mentions)
P21waf1, by Santa Cruz Biotechnology (1 mentions)
Horseradish conjugated secondary antibodies, by GE Healthcare (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Tubulin, by Santa Cruz Biotechnology (1 mentions)
Blimp1, by Santa Cruz Biotechnology (1 mentions)
Protein g sepharose fast flow beads, by GE Healthcare (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Anti trx1, by Cell Signaling Technology (1 mentions)
Lysis buffer, by Thermo Fisher Scientific (1 mentions)
Anti albumin, by Cell Signaling Technology (1 mentions)
Anti sirtuin 1 sirt1, by Cell Signaling Technology (1 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
Anti hdac6, by Abcam (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by GE Healthcare (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Crescendo western hrp substrate, by Merck Group (1 mentions)
Phenylmethanesulfonyl fluoride, by Merck Group (1 mentions)
7 protocols using stripping buffer
1
Western Blot and Immunoprecipitation Protocol
2
Immunoblotting Protein Expression Analysis
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Immunoblotting was performed to assess protein expression in response to the different treatments, as previously described [31 (link)]. Briefly, control and treated HuREC were lysed using radioimmunoprecipitation assay buffer lysis buffer containing 1% phosphatase and protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Fifty micrograms of each protein sample was electrophoresed on SDS-PAGE and transferred onto a Polyvinylidene difluoride (PVDF) membrane. The membrane was then blocked using 5% skim milk and incubated with the following primary antibodies: SIRT1 (1:1000; Cell Signaling) and p16INK4a, TrxR2 (1:1000; Abcam, Cambridge, MA), p21Waf1 and SOD2 (1:500; Santa Cruz Biotech, Dallas, TX, USA), and corresponding secondary horseradish-conjugated antibodies (GE Healthcare, Pittsburg, PA, USA). After immunoblotting, the membranes were stripped using stripping buffer (Bio-Rad) and re-probed with anti-β-actin antibody (1:3000; Sigma-Aldrich, St. Louis, MO, USA). Chemiluminescence-based assay was used for band detection (ThermoFisher, Waltham, MA, USA). Scanned images of blots were used to quantify protein expression using NIH ImageJ software (http://rsb.info.nih.gov/ij/ ).
3
Immunoprecipitation and Western Blot Analysis
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Transient transfection was performed using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions, in 60-mm dishes and using 2–3 µg of total DNA per transfection. 2 d after transfection, cells were lysed in 1× Nonidet P-40 lysis buffer and freshly added protease inhibitor cocktail. The cell lysates were mixed with antibodies (1 µg) for 2 h, followed by the addition of 30 µl of fast flow protein G–Sepharose beads (GE Healthcare) for an additional 2 h at 4°C. Immunoprecipitates were washed four times with Nonidet P-40 lysis buffer and boiled in 20 µl of 2× Laemmli’s buffer. Samples were subjected to 8–12% SDS-PAGE analysis and electro-transferred onto polyvinylidene difluoride membranes (Millipore). Membranes were probed with the indicated primary antibodies against Hrd1 (Sigma-Aldrich), BLIMP1 (Santa Cruz Biotechnology, Inc.), and Tubulin (Santa Cruz Biotechnology, Inc.), followed by horseradish peroxidase–conjugated secondary antibodies. Membranes were then washed and visualized with an enhanced chemiluminescence detection system (ECL; GE Healthcare). When necessary, membranes were stripped by incubation in stripping buffer (Bio-Rad Laboratories), washed, and then reprobed with other antibodies as indicated.
4
Co-Immunoprecipitation and Western Blotting
5
Retinal Protein Expression Analysis
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Retinal tissue was homogenized in lysis buffer (ThermoFisher, Waltham, MA, USA) containing 1% phosphatase and protease inhibitor cocktail (Sigma-Aldrich). Protein concentration was measured using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s recommendation. Proteins from whole rat retinal tissue and HuREC lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membrane. The membrane was blocked using 5% skim milk and incubated with the following primary antibodies: anti-HDAC6 (Abcam, Cambridge, MA, USA), anti-Trx-1, anti-sirtuin 1 (SIRT1) and anti-albumin (all from Cell Signaling Technology). After incubation with horseradish peroxidase–conjugated secondary antibody (GE Healthcare, Pittsburg, PA, USA), bands were detected using the enzymatic chemiluminescence reagent, ECL (GE Healthcare). Subsequently, the membranes were stripped using stripping buffer (Bio-Rad) and re-probed with anti-β-actin antibody (Sigma-Aldrich) to assess equal loading. Scanned images of blots were used to quantify protein expression using NIH ImageJ software (http://rsb.info.nih.gov/ij/ ).
6
Co-IP and Western Blotting of Transfected Cells
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Co-IP and western blotting were performed as described [20 (link)]. Transiently transfected HCT116 cells were washed with ice-cold phosphate-buffered saline (PBS), resuspended in RIPA lysis buffer with protease inhibitor and incubated on ice for 15 min. Insoluble fractions were removed by centrifugation (15 000 g, 15 min). Supernatants were pre-cleaned with protein G-sepharose at 4 °C for 15 min and then incubated with the indicated antibody (1 μg ml−1) for 1 h followed by incubation with protein G-sepharose beads for 2 additional hours. The protein G Sepharose beads were washed four times with lysis buffer, dissolved with 4× loading buffer and boiled for 5 min. Supernatants were subjected to SDS–PAGE and transferred to nitrocellulose membrane. After blocking with 5% (w/v) skim milk in Tris-buffered saline containing 0.1% Tween 20, the membrane was incubated overnight at 4 °C with the indicated primary antibodies followed by horseradish peroxidase-conjugated secondary antibody. Membranes were then washed and visualized with enhanced chemiluminescence. When necessary, membranes were stripped using stripping buffer (Bio-Rad, Hercules, CA, USA) and reprobed with corresponding antibodies.
7
Protein Extraction and Western Blot Analysis
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Cells or homogenized xenograft specimens were lysed on ice for one hour in radio-immunoprecipitation assay (RIPA) buffer supplemented with protease inhibitors (Sigma Aldrich), phosphatase inhibitors (Sigma Aldrich), and phenyl-methane-sulfonyl-fluoride (Sigma Aldrich). Lysates were centrifuged at 14,000 rpm for 1 h at 4 °C. The Pierce BCA Protein Assay reagent (Thermo Fisher Scientific) was used to determine protein concentrations. Lysates were then separated by electrophoresis on sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gels. Precision Plus Protein Kaleidoscope Standards (Bio-Rad) were used to confirm the expected size of the target proteins. Antibodies were used according to the manufacturers’ recommended conditions. Luminata Classico or Crescendo Western HRP Substrate (EMD Millipore) were used to develop the immunoblots. Blots were stripped with stripping buffer (Bio-Rad) at 65 °C for 20 min and then re-probed with antibodies to proteins of interest. GAPDH, β-actin, or vinculin were used to confirm equal protein loading.
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