Total proteins were separated with radioimmunoprecipitation assay buffer (Biosharp, Hefei, China) containing 1 mM phenylmethylsulfonyl fluoride and a phosphatase inhibitor cocktail (MedChemExpress, NJ, USA). After centrifugation at 12,000 rpm for 15 min at 4°C, the concentration of proteins was determined with the BCA protein assay kit (AMEKO, Shanghai, China). The proteins (30 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis using 10% gels (Epizyme, Shanghai, China) and transferred to polyvinylidene difluoride membranes. We cut the membranes horizontally into strips for detecting proteins with different molecular weights. The membranes were blocked with blocking buffer (Biosharp) for 1–4 h and incubated with primary antibody for 12 h at 4°C. Next, the membranes were incubated with secondary antibody for 1 h at room temperature, followed by the detection of target proteins with an ultra-sensitive ECL chemiluminescence kit (NCM Biotech, Suzhou, China) and the ImageQuant LAS 500 system (GE Healthcare, Fairfield, CT, USA). The intensity of each band was quantified by ImageJ software (Bethesda, MD, USA) and normalized using β-actin.
Blocking buffer
Manufactured by Biosharp
Sourced in China, United States
Blocking buffer is a solution used in various immunoassays, such as Western blotting and ELISA, to reduce non-specific binding of antibodies or other proteins to the solid support or membrane. It typically contains a combination of proteins, detergents, and other additives that help to block any unoccupied binding sites, preventing unwanted interactions and improving the specificity of the assay.
Automatically generated - may contain errors
Lab products found in correlation
Phosphatase inhibitor cocktail, by MedChemExpress (1 mentions)
Radioimmunoprecipitation assay buffer, by Biosharp (1 mentions)
Imagequant las 500 system, by GE Healthcare (1 mentions)
Anti notch1, by Abcam (1 mentions)
Anti hes1, by Abcam (1 mentions)
Anti hey1, by Abcam (1 mentions)
Ripa containing protease inhibitors, by Beyotime (1 mentions)
Anti β actin, by Affinity Biosciences (1 mentions)
Enhanced chemiluminescence system, by Bio-Rad (1 mentions)
Bca protein assay kit, by Solarbio (1 mentions)
Imagequant las 500, by GE Healthcare (1 mentions)
Page gel fast preparation kit, by Ipsen (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
4 protocols using blocking buffer
1
Quantitative Protein Analysis by Western Blot
2
Western Blot Analysis of Notch Signaling Pathway
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Tissues or cells were lysed with RIPA containing protease inhibitors (Beyotime, China) on ice for 30 min. Samples were separated on SDS-PAGE gels and blotted onto PVDF membranes. After being blocked with blocking buffer (Biosharp, China) for 1 hour, the PVDF membranes were incubated with the following diluted primary antibodies at 4°C overnight: anti-CENPU (Affinity, China, 1 : 1000), anti-Notch1 (Abcam, USA, 1 : 1000), anti-Hes1 (Abcam, USA, 1 : 1000), anti-Hey1 (Abcam, China, 1 : 1000), and anti-β-actin (Affinity, China, 1 : 2000). Then, the PVDF membranes were incubated with specific horseradish peroxidase- (HRP-) conjugated IgG secondary antibodies (Affinity, 1 : 2000). The protein bands on the PVDF membrane were visualized by using a Tanon 5200 Imaging System.
3
Quantitative Immunoblotting Protocol for Protein Analysis
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We performed immunoblotting experiments as described previously [4 (link)]. The issue to be tested in radioimmunoprecipitation assay (RIPA) buffer and centrifuged at 12,000 rpm for 15 min at 4 ℃. The supernatant was collected and the total protein content was determined by bicinchoninic acid (BCA) Protein Assay Kit (Solarbio). The protein was separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to polyvinylidene fluoride (PVDF) membrane. The membrane was sealed with Blocking Buffer (Biosharp) for 30 min. the membranes were incubated with primary antibodies at appropriate dilutions in PBS/Tween (PBST) overnight at 4 ℃. subsequently, after washing with PBS/Tween (PBST) for 3 × 5 min, the membrane was incubated with secondary antibody at 37 ℃ for 2 h. After washing, the results were visualized using an enhanced chemiluminescence system (Bio Rad).
4
Quantitative Protein Analysis of Nucleus Pulposus Cells
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The total protein of nucleus pulposus cells was lysed and extracted using radioimmunoprecipitation buffer (RIPA) (Biosharp, China) (100:1) containing phenylmethanesulfonyl fluoride (PMSF). The protein sample was centrifuged at 4°C, 12,000 rpm for 15 min, and then the protein concentration was determined using the BCA protein detection kit (AMEKO, Shanghai, China). Prepare the gel according to the instructions of the PAGE Gel Fast Preparation Kit (Epizyme, Shanghai, China), separate the protein samples, and then transfer them to the PVDF membrane (Bio-Rad, Hercules, CA, United States). And use blocking buffer (Biosharp, Hefei, China) to seal at room temperature for 50 min. Incubate the PVDF membrane with the primary antibody overnight at 4°C, and then incubate with the secondary antibody for 1 h at room temperature. Ultra-sensitive ECL chemiluminescence kit (NCM Biotech, Suzhou, China) and ImageQuant LAS 500 (GE Health Care, Fairfield, CT, United States) were used to detect antibody reactivity. ImageJ software quantitatively analyzes the band intensity.
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