The cells were lysed with RIPA buffer (Boster, China) supplemented with protease/phosphatase inhibitor Cocktail (Boster, China). Proteins from the lysate were separated by electrophoresis using 10–12% polyacrylamide gels and electrotransferred to PVDF membranes (Bio-Rad). After blocking with 5% skimmed milk, membranes were incubated with various primary antibodies. Then membranes were incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibody. Specific proteins were detected with EZ-ECL Kit (Biological Industries).
Protease phosphatase inhibitor cocktail
Manufactured by Boster Bio
Sourced in China
Protease/phosphatase inhibitor Cocktail is a laboratory reagent designed to inhibit the activity of proteases and phosphatases in biological samples. It is used to preserve the integrity of proteins and their post-translational modifications during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Bio-Rad (4 mentions)
Ripa buffer, by Boster Bio (3 mentions)
Ez ecl kit, by Sartorius (3 mentions)
Bradford protein assay kit, by Bio-Rad (3 mentions)
Ecl kit, by Bio-Rad (1 mentions)
Enhanced ecl kit, by Thermo Fisher Scientific (1 mentions)
Anti lamin b1, by Abcam (1 mentions)
Anti p65, by Santa Cruz Biotechnology (1 mentions)
5 protocols using protease phosphatase inhibitor cocktail
1
Western Blot Analysis of Proteins
2
Western Blot Analysis of Protein Expression
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Cells or tumor tissues were lysed in protein lysis buffer with protease/phosphatase inhibitor cocktail (Boster, China), and centrifuged at 12,000 rpm for 10 min at 4 °C to remove debris. The protein concentrations were measured by using the Bradford protein assay kit (Bio-Rad, Herculer, CA). Protein samples were subjected to electrophoresis on 10 % or 12 % polyacrylamide gels, and the separated proteins were transferred onto PVDF membranes (Bio-Rad). The membrane was incubated with fresh 5 % skimmed milk in TBST for 1.5 h at room temperature before being incubated overnight at 4 °C with indicated primary antibodies. Following washing the membrane with TBST three times, the membranes were further incubated in HRP-linked secondary antibodies for 1 h at room temperature. Finally, ECL kit was employed to visualize the protein bands (Bio-Rad, Hercules, CA).
3
Western Blot Analysis of Rat Chondrocytes
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Rat chondrocytes were collected and washed with phosphate buffered saline (PBS). Next, the cells were lysed with RIPA buffer containing 1% protease/phosphatase inhibitor cocktail (Boster). The protein concentration of isolated cell lysis solution was determined by bicinchoninic acid (BCA) kit. Afterward, 25 μg protein samples were separated on 8–12% SDS–polyacrylamide gels and transferred to PVDF membranes using a Bio-Rad system. The membranes were blocked with 5% BSA for 1 h at room temperature and then incubated with primary antibodies overnight at 4°C. Subsequently, the membranes were washed with TBST and incubated with corresponding secondary antibodies for 1 h at room temperature. Finally, enhanced ECL kit (Thermo Fisher Scientific, United States) was used to visualize the blots. The relative protein expression was calculated by ImageJ software compared to internal control.
4
Western Blot Protein Analysis
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The cells were lysed with RIPA buffer (Boster, China) containing protease/phosphatase inhibitor Cocktail (Boster, China). Total proteins from cultured cells were isolated and the concentrations were measured by the Bradford protein assay kit (Bio-Rad, Hercules, CA). After 10–12% SDS-PAGE, the proteins were electrotransferred to PVDF membrane (Bio-Rad). Then, membranes were blocked with 5% Nonfat Milk in Tris Buffered Saline (TBS) with 1‰Tween 20 (1 × TBST) for 90 min at room temperature. Next, the membranes were incubated with primary antibody in TBST overnight at 4 °C, followed by incubation with appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (x 4000) for 1 h at room temperature. Specific proteins were detected with EZ-ECL Kit (Biological Industries). Primary antibodies from Cell Signaling Technology, Proteintech (Anti-Lamin B1) and abcam (Anti-QKI-5) were diluted 1000 times. Primary antibody from Santa Cruz Biotechnology (Anti-p65) was diluted 500 times.
5
Protein Extraction and Western Blot
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The cells
were lysed with RIPA buffer (Boster, China) containing protease/phosphatase
inhibitor cocktail (Boster, China). Total proteins from cultured cells
were isolated, and the concentrations were measured by the Bradford
protein assay kit (Bio-Rad, Hercules, CA). After 10–12% SDS-PAGE,
the proteins were electrotransferred to the PVDF membrane (Bio-Rad).
Then, membranes were blocked with 5% nonfat milk in Tris-buffered
saline (TBS) with 1% Tween-20 (1× TBST) for 90 min at room temperature.
Next, the membranes were incubated with a primary antibody in TBST
overnight at 4 °C, followed by incubation with an appropriate
horseradish peroxidase (HRP)-conjugated secondary antibody (×4000)
for 1 h at room temperature. Specific proteins were detected with
an EZ-ECL Kit (Biological Industries). Primary antibodies were obtained
from Cell Signaling Technology.
were lysed with RIPA buffer (Boster, China) containing protease/phosphatase
inhibitor cocktail (Boster, China). Total proteins from cultured cells
were isolated, and the concentrations were measured by the Bradford
protein assay kit (Bio-Rad, Hercules, CA). After 10–12% SDS-PAGE,
the proteins were electrotransferred to the PVDF membrane (Bio-Rad).
Then, membranes were blocked with 5% nonfat milk in Tris-buffered
saline (TBS) with 1% Tween-20 (1× TBST) for 90 min at room temperature.
Next, the membranes were incubated with a primary antibody in TBST
overnight at 4 °C, followed by incubation with an appropriate
horseradish peroxidase (HRP)-conjugated secondary antibody (×4000)
for 1 h at room temperature. Specific proteins were detected with
an EZ-ECL Kit (Biological Industries). Primary antibodies were obtained
from Cell Signaling Technology.
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