Total protein was extracted from lung samples or cell samples using Radio-Immunoprecipitation Assay and PMSF reagent (Beyotime, Beijing, China). Protein lysates were separated based on their molecular weight on SDS/PAGE gels and transferred onto a Polyvinylidene Fluoride (PVDF, Millipore) membrane. The membrane was blocked with nonfat-milk (5%) for 2 hours at room temperature and incubated with ant-TCF4 antibody, anti-bHLH antibody and anti-β-actin antibody at 4°C overnight. Then first antibodies were removed and TBST wash membrane using TBST. Membrane were incubated with the secondary antibody for 2 hours at room temperature. The bound antibodies were detected using enhanced chemiluminescence with β-actin used as a control.
Pmsf reagent
Manufactured by Beyotime
Sourced in China
PMSF reagent is a protease inhibitor commonly used in biochemical research. It functions by irreversibly inhibiting serine proteases, which are a class of enzymes involved in the breakdown of proteins. The core purpose of PMSF is to prevent proteolytic degradation of target proteins during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene fluoride (pvdf), by Merck Group (2 mentions)
Ripa reagent, by Beyotime (2 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
Ecl solution, by Merck Group (1 mentions)
Anti p62 p0067, by Merck Group (1 mentions)
Ab6721, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ab183218, by Abcam (1 mentions)
Ab187671, by Abcam (1 mentions)
Ab9485, by Abcam (1 mentions)
Ab9722, by Abcam (1 mentions)
Beyoecl moon chemiluminescence kit, by Beyotime (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Beyotime (1 mentions)
Bovine serum albumin (bsa), by Solarbio (1 mentions)
Phosphatase inhibitor cocktail, by MedChemExpress (1 mentions)
Amersham imager, by Cytiva (1 mentions)
Ab192890, by Abcam (1 mentions)
Ab237751, by Abcam (1 mentions)
Anti p62 antibody p0067, by Merck Group (1 mentions)
Ab8226, by Abcam (1 mentions)
6 protocols using pmsf reagent
1
Protein Expression Analysis in Lung Samples
2
Corneal Cells Autophagy and Inflammation
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The corneal stromal cells or mouse corneal tissues were harvested and lysed with RIPA reagent (Beyotime, Shanghai, China) supplemented with 1% Phenylmethylsulfonyl Fluoride (PMSF) reagent (Beyotime). Recombinant proteins (20 µg) were electrophoresed on 12.5% SDS-PAGE gels, transferred electrophoretically to PVDF membranes (Millipore, Boston, MA, USA), and blocked with 5% skimmed milk for 60 minutes. Afterward, the proteins were incubated with the corresponding primary antibodies at 4°C for 12 hours. The antibodies were as follows: anti-P62 (P0067; Sigma-Aldrich), anti-LC3B (ab192890; Abcam, Cambridge, MA, USA), anti-ATG16L1 (ab187671; Abcam), anti–TNF-α (ab183218; Abcam), anti–IL-1β (ab9722; Abcam), and anti-GAPDH (ab9485; Abcam) antibodies. On the second day, membranes were washed with TBS+Tween (TBST) thrice, incubated with the secondly antibodies horseradish peroxidase (HRP) (ab6721, Abcam) for 60 minutes, and washed again. Finally, the protein bands were visualized with ECL solution (Millipore).
3
Western Blotting Protein Quantification
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Total protein was extracted using radio-immunoprecipitation assay lysis buffer (Beyotime, Shanghai, China) containing 1% PMSF reagent (Beyotime, Shanghai, China) and a 1% phosphatase inhibitor cocktail (MedChemExpress, Shanghai, China). Denatured total proteins were electrophoresed on a 12% polyacrylamide gel and transferred onto polyvinylidene fluoride membranes. Blocking was performed for 1 h in 5% bovine serum albumin (Solarbio, Beijing, China), and membranes were incubated overnight at 4 °C with the indicated rabbit anti-human antibodies (1:3000). Subsequently, the membranes were incubated with HRP-conjugated goat anti-rabbit secondary antibodies (1:5000) for 1 h. The blots were visualised using the BeyoECL Moon chemiluminescence kit (Beyotime, Shanghai, China) on the Amersham Imager 680 imager. Grey values were analysed using the ImageJ software. Band densities were normalised to that of β-actin or GAPDH.
4
Autophagy Protein Expression in Corneal Epithelium
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The TG tissues were harvested on day 5 after corneal epithelial debridement and lysed with RIPA reagent (Beyotime, Shanghai, China) supplemented with 1% Phenylmethylsulfonyl Fluoride (PMSF) reagent (Beyotime). Protein samples were electrophoresed on Sodium dodecyl sulfate–polyacrylamide gels, transferred to polyvinylidene fluoride membranes, and blocked with skim milk. Afterward, the membranes were incubated in solutions of the corresponding primary antibodies and horseradish peroxidase-conjugated secondary antibodies. Eventually, Enhanced chemiluminescence was used to visualize proteins bands. At least three independent experiments were performed. The primary antibodies used for the study were as follows: anti-p62 antibody (p0067; Sigma-Aldrich, USA), anti-LC3B (ab192890; Abcam, USA), anti-ATG4D (ab237751; Abcam, USA), and anti-β-actin (ab8226; Abcam, USA).
5
Exosome Protein Analysis by Western Blot
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Briefly, DETCs-derived exosomes and 3T3 cells were lysed in a RIPA lysis buffer (Beyotime) supplementing with a protease inhibitor and PMSF reagent (Beyotime) for 30 min on ice. The extracted protein was quantified by a BCA protein assay kit (Thermo Fisher Scientific). Then, a total of 30 µg protein samples were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were then incubated with primary antibodies overnight at 4 °C using anti-mouse Calnexin (1:1000, ab133615, Abcam), anti-mouse CD63 (1:1000, ab217345, Abcam) or anti-mouse TSG101 (1:1000, ab125011, Abcam). Subsequently, the membranes were incubated with HRP-labeled secondary antibodies (Sungene Biotech) for 60 min at room temperature. Chemiluminescence reagents and the ChemiDoc™ XRS western blot detection system (Bio-Rad) were used to detect the proteins of interest.
6
Quantification of Protein Expression in Cancer
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Total protein was extracted from cancer tissue and paracancerous tissue samples or cell samples using Radio-Immunoprecipitation Assay (RIPA) and PMSF reagent (Beyotime, Beijing, China). Protein lysates were separated based on their molecular weight on SDS/PAGE gels and transferred onto a Polyvinylidene Fluoride (PVDF, Millipore) membrane. The membrane was blocked with non-fat-milk (5%) for 2 h at room temperature and incubated with ant-EIF3D antibody, anti-GRP78 antibody and anti-β-actin antibody at 4 °C overnight. Then first antibodies were removed and TBST wash membrane using TBST. Membrane were incubated with the secondary antibody for 2 hours at room temperature. The bound antibodies were detected using enhanced chemiluminescence (ECL) with β-actin used as a control.
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