Cells were lysed using 200 µl lysis buffer NP40 (Beyotime Institute of Biotechnology). The concentration of the samples was determined by BCA protein quantitative kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Proteins were separated using 10% SDS-PAGE gels and transferred onto a PVDF membrane (EMD Millipore, Billerica, MA, USA). After being blocked with 5% non-skimmed milk at room temperature for 2 h, the membrane was incubated with primary antibodies as listed: cleaved caspase-3 (ab32042, 1:500; Abcam, Cambridge, UK), Fas (ab82419, 1:1,000), Fasl (ab15285, 1 ug/ml), Bax (ab32503,1:2,000), Bcl-2 (ab32124, 1:1,000), p-AKT (ab812831:5,000), AKT 1/2 (ab182729, 1:5,000), p-eNOS (ab184154, 1:1,000), eNOS (ab76198, 1:1,000), and GAPDH (ab8245, 1:5,000), at 4°C overnight. Then, horseradish peroxidase-conjugated secondary antibody (ab6721, 1:4,000) was added and maintained at room temperature for 1 h. The blot bands were developed using BeyoECL Star (Beyotime Institute of Biotechnology). The gray density was calculated with Quantity One software version 4.6 (Bio-Rad Laboratories, Inc.).
Bca protein quantitative kit
Manufactured by Bio-Rad
Sourced in United States
The BCA Protein Quantitative Kit is a laboratory tool used for the colorimetric detection and quantitation of total protein concentration in a sample. It is based on the bicinchoninic acid (BCA) method, which combines the reduction of Cu2+ to Cu+ by proteins in an alkaline medium with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu+) using a reagent containing bicinchoninic acid.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (1 mentions)
Quantity one software version 4, by Bio-Rad (1 mentions)
Ab32042, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Ab82419, by Abcam (1 mentions)
Ab184154, by Abcam (1 mentions)
Ab15285, by Abcam (1 mentions)
Ab6721, by Beyotime (1 mentions)
Beyoecl star, by Beyotime (1 mentions)
Ab32124, by Abcam (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti eif4e, by Cell Signaling Technology (1 mentions)
Anti erk1 2, by Cell Signaling Technology (1 mentions)
Anti s6k, by Cell Signaling Technology (1 mentions)
Anti mtor, by Cell Signaling Technology (1 mentions)
Anti pi3k, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
2 protocols using bca protein quantitative kit
1
Western Blot Analysis of Apoptosis Markers
2
PI3K/Akt/mTOR Signaling Pathway Analysis
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Cells were harvested in RIPA lysis buffer with 1% PMSF (Beyotime, China), which was centrifuged at 10,000 × g for 5 min at 4 °C, the supernatant was collected. And protein content was measured using BCA protein quantitative kit (Bio-Rad, USA). Protein expression was determined by the western blot as described 10 (link). The following primary antibodies were used: anti-PI3K (Cell Signaling Technology, USA), anti-Akt (Cell Signaling Technology, USA), anti-mTOR (Cell Signaling Technology, USA), anti-S6k (Cell Signaling Technology, USA), anti-Eif4e (Cell Signaling Technology, USA), anti-Erk1/2 (Cell Signaling Technology, USA), anti-Mek1 (upstate, USA) and anti-GAPDH (Proteintech, China).
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