Cellular protein was harvested by using RIPA buffer (Sigma) and quantified by using a BCA protein assay (GenDEPOT). Equal protein amounts were resolved by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech). The membranes were incubated overnight with specific antibodies against α-SMA (Abcam), pSMAD2 (Thermofisher), SMAD2 (Thermofisher) and anti-GAPDH (Abcam). The next day, the membranes were washed, incubated with a peroxidase-linked IgG (Abcam), and visualized by using an ECL kit (WESTSAVE Gold).
Bca protein assay
Manufactured by GenDEPOT
Sourced in United States
The BCA protein assay is a colorimetric detection and quantification method used to measure the total protein concentration in a sample. It utilizes the bicinchoninic acid (BCA) reagent to produce a purple-colored reaction, which can be measured spectrophotometrically. The assay is a simple, accurate, and sensitive way to determine the total protein content in a variety of sample types.
Automatically generated - may contain errors
Lab products found in correlation
P smad2, by Thermo Fisher Scientific (1 mentions)
α sma, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Cytiva (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Peroxidase linked igg, by Abcam (1 mentions)
Smad2, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Abcam (1 mentions)
2 protocols using bca protein assay
1
Western Blot Analysis of Cell Proteins
2
Quantifying Intracellular Cholesterol Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
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HepG2 cells and liver tissues were treated with chloroform/isopropanol (2:1) for 30 min at room temperature, and then the lipid-extracted solvent was transferred to test tubes. The organic solvent was removed using a vacuum centrifuge, and the lipids were re-suspended in 95% ethanol. The intracellular cholesterol level was quantified with a cholesterol/cholesteryl ester kit (Abcam, Cambridge, UK) according to the manufacturer's instructions. After lipid extraction, cells were lysed with RIPA buffer and centrifuged at 13,000× g for 10 min to collect the supernatant for the measurement of cellular protein concentrations. Lipid levels were normalized to the total cellular protein concentration as determined using the bicinchoninic acid (BCA) protein assay (GenDEPOT, Katy, TX, USA).
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