Cells were washed twice with PBS and total protein was extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology) supplemented with PMSF protein inhibitor (Beyotime Institute of Biotechnology). The protein concentration was quantified using a standard BCA protein assay and 30 µg of protein/lane was separated via 10% SDS-PAGE. The separated proteins were subsequently transferred onto PVDF membranes (MilliporeSigma) and blocked with 5% non-fat milk at 26˚C for 2 h. The membranes were then incubated overnight at 4˚C with the following primary antibodies: Rabbit anti-COPS7A (1:1,000; cat. no. ab124705; Abcam), rabbit anti-CDK2 (1:1,000; cat. no. ab32147; Abcam), rabbit anti-CDK4 (1:1,000; cat. no. ab108357; Abcam), rabbit anti-cyclin D2 (1:1,000; cat. no. ab230883; Abcam) and rabbit anti-GAPDH (1:2,500; cat. no. ab9485; Abcam). Following the primary antibody incubation, the membranes were incubated with a goat anti-rabbit IgG H&L secondary antibody (1:5,000; cat. no. ab6721; Abcam) at room temperature for 2 h according to the protocol. The protein bands were visualized using an ECL system (Thermo Fisher Scientific, Inc.). GAPDH was used as the internal loading control.
Pmsf protein inhibitor
Manufactured by Beyotime
Sourced in China
PMSF (Phenylmethanesulfonyl fluoride) is a protease inhibitor commonly used in the laboratory for the preservation of proteins. It functions by irreversibly inhibiting a wide range of serine proteases, preventing the degradation of proteins during extraction and purification processes.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Ecl system, by Thermo Fisher Scientific (1 mentions)
Rabbit anti cdk2, by Abcam (1 mentions)
Rabbit anti cdk4, by Abcam (1 mentions)
Rabbit anti gapdh, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Rabbit anti fgf21 primary antibody, by Abcam (1 mentions)
Bcl 2, by Beyotime (1 mentions)
Hsp90, by Beyotime (1 mentions)
Hrp coupled secondary antibody, by Beyotime (1 mentions)
0.2 μm pvdf membrane, by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
3 protocols using pmsf protein inhibitor
1
Western Blot Analysis of Cell Cycle Regulators
2
Hepatic Protein Expression Analysis
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Hepatic lysate was isolated using radioimmunoprecipitation assay (RIPA) buffer supplemented 1 mM phenylmethylsulfonyl fluoride (PMSF) protein inhibitor (Beyotime Biotechnology, China). Forty micrograms of protein in each lane was electrophoresed with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to 0.2-μm polyvinyldene fluoride (PVDF) membranes (Millipore, MA, USA). After blocking with 5% non-fat milk solution (Yili Industrial Group Co., Ltd, China) for 1 h, the membrane was incubated with diluted rabbit-anti-FGF21 primary antibody (1:1000, Abcam, UK), rabbit-anti-PARP, and cleaved PARP (1:1000, Cell Signaling Technology, USA) overnight at 4°C, developed using a horseradish peroxidase (HRP)-coupled secondary antibody (Beyotime Biotechnology) for 1 h. After adding the chemiluminescent reagent, the membrane was imaged and captured using a gel imager (BIO-RAD, USA). Internal α-tubulin protein (Beyotime Biotechnology) was used as a housekeeping control for target protein normalization [23 (link)–24 (link)].
3
Western Blot Analysis of Mouse Liver Proteins
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Mouse liver protein was extracted by using RIPA buffer plus 1mM PMSF protein inhibitor (Beyotime Biotechnology, China). And 40μg protein/lane (liver tissue) and 10μg protein/lane (cell strain) were separated with 10% SDS-PAGE, and then was blotted to 0.2μm PVDF membrane (Millipore, MA, USA). After being blocked with 5% non-fat milk buffer (Yili Industrial Group Co., Ltd, China) for 1 h at room temperature, membrane was incubated with diluted Hsp90 (1:500, Beyotime, China), AKR7A (1:1000, Santa Cruz, USA), Bcl-2 (1:500, Beyotime, China), p44/42MAPK (1:1000, Beyotime, China), Actin (1:500, Beyotime, China) primary antibodies overnight at 4 °C, followed by HRP-coupled secondary antibody (1:2000, Beyotime, China) for 1h at room temperature. After incubating with chemiluminescent agent, the membrane was developed and imaged under Imager System (Bio-Rad, USA) [16] .
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