Cells were washed with PBS and scraped immediately in RIPA lysis buffer (Santa Cruz Biotechnology, Inc.) with 200 mM PMSF, 100 mM sodium orthovanadate and a protease inhibitor cocktail. The protein concentration was determined using the BCA assay protein quantitation kit (Interchim). Cell extracts were heated at 95°C for 5 min, separated (50 µg protein/lane) by 10% SDS-PAGE in reducing conditions (5% 2β-mercaptoethanol), and transferred to PVDF membranes (Bio-Rad Laboratories, Inc.). The membranes were blocked in Tris-buffered saline with 0.1% Tween-20 and 5% non-fat dry milk, and probed with the relevant primary antibodies at RT for 1 h. Following washing with PBS-Tween 0.01%, peroxidase-conjugated IgG secondary antibodies were added (1:10,000) at RT for 1 h. After washing, antibody-antigen interactions were detected using Immobilon® ECL Ultra Western HRP Substrate (Merck KGaA). To verify equal loading, immunoblots were also probed with an anti-GAPDH monoclonal antibody diluted at 1:1,000 (cat. no. 8884; Cell Signaling Technology, Inc.).
Bca assay protein quantitation kit
Manufactured by Interchim
The BCA assay protein quantitation kit is a colorimetric-based method for determining the total protein concentration in a sample. The kit utilizes the bicinchoninic acid (BCA) reaction, which produces a purple-colored complex when proteins react with an alkaline copper sulfate solution. The intensity of the color is proportional to the protein concentration, allowing for accurate quantification.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Bio-Rad (2 mentions)
Ripa lysis buffer, by Santa Cruz Biotechnology (2 mentions)
Immobilon ecl ultra western hrp substrate, by Merck Group (1 mentions)
Chemiluminescent substrate, by Merck Group (1 mentions)
Anti gapdh monoclonal antibody, by Cell Signaling Technology (1 mentions)
G box ichemi, by Syngene (1 mentions)
2 protocols using bca assay protein quantitation kit
1
Western Blot Protein Analysis Protocol
2
Western Blot Analysis of Protein Extracts
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Cells were washed with PBS and scraped immediately in RIPA lysis buffer (Santa Cruz) that included 200 mM PMSF solution, 100 mM sodium orthovanadate solution, and protease inhibitor cocktail. The protein concentration was determined using the BCA assay protein quantitation kit (Interchim). Cell extracts were heated at 95 °C for 5 min, separated (50 μg proteins/line) on 10% SDS-PAGE in reducing conditions (5% 2β-mercaptoethanol), and transferred to PVDF membranes (Biorad). Membranes were saturated in Tris-buffered saline, containing 0.1% Tween-20 and 5% non-fat dry milk, and probed with the relevant primary antibodies at room temperature for 1 h. After washing, peroxidase-conjugated IgG secondary antibodies were added (1/10,000) at room temperature for 1 h. After washing, antibody-antigen interactions were detected using a chemiluminescent substrate (Merck). To verify equal loading, immunoblots were also probed with an anti-GAPDH monoclonal antibody (Cell Signaling, Cat# 8884). Membrane exposition was performed using the G:BOX iChemi (Syngene) and did not exceeded 5 min. Image acquisition was performed using the GeneSys and its processing using ImageJ software.
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