Total proteins were extracted from cultured cells by RIPA buffer. The results showed that the BCA detection method (Beyotime, Nantong, Jiangsu, China) quantitatively detected all proteins. The protein samples separated by PAGE (polyacrylamide gel electrophoresis) were transferred to the PVDF membranes (EMD Millipore, Billerica, Massachusetts, USA). The PVDF membranes were blocked in 5% skim milk for 1 h, then incubated with specific antibodies overnight at 4 °C, and finally incubated with the secondary antibodies for 2 h at room temperature. The membranes were visualized using enhanced chemiluminescence solution (Pierce Biotechnology, Inc. USA). The experimental results were analyzed by ImageJ analysis software. The following antibodies were recorded: anti-GAPDH, anti-FLAG-tag, anti-HIF1α, anti-YTHDF2 (Proteintech, Wuhan, Hubei, China), anti-AKT, anti-Phospho-AKT (Ser473), anti-ERK1/2, anti-Phospho-ERK1/2 (Thr202/Tyr204, Cell Signaling Technologies, Danvers, MA, USA), anti-Phospho-mTOR (Ser2448), anti-mTOR, anti-E-cadherin, anti-N-cadherin, anti-Vimentin, anti-Snail1, anti-METTL3, anti-METTL14.
Bca detection method
Manufactured by Beyotime
Sourced in China
The BCA detection method is a colorimetric assay used to quantify the total protein concentration in a sample. It is based on the reduction of copper ions by proteins in an alkaline environment, which results in the formation of a purple-colored complex that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Enhanced chemiluminescence solution, by Thermo Fisher Scientific (2 mentions)
Anti phospho mtor, by Cell Signaling Technology (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti n cadherin, by Cell Signaling Technology (2 mentions)
Anti e cadherin, by Cell Signaling Technology (2 mentions)
Anti snail1, by Cell Signaling Technology (2 mentions)
Anti phospho erk1 2 thr202 tyr204, by Cell Signaling Technology (2 mentions)
Anti mtor, by Cell Signaling Technology (2 mentions)
Anti hif 1α, by Proteintech (2 mentions)
Anti ythdf2, by Proteintech (2 mentions)
Anti vimentin, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Proteintech (2 mentions)
Anti flag tag, by Proteintech (2 mentions)
2 protocols using bca detection method
1
Comprehensive Protein Analysis Protocol
2
Comprehensive Protein Analysis Protocol
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Total proteins were extracted from cultured cells by RIPA buffer. The results showed that the BCA detection method (Beyotime, Nantong, Jiangsu, China) quantitatively detected all proteins. The protein samples separated by PAGE (polyacrylamide gel electrophoresis) were transferred to the PVDF membranes (EMD Millipore, Billerica, Massachusetts, USA). The PVDF membranes were blocked in 5% skim milk for 1 hour, then incubated with speci c antibodies overnight at 4°C, and nally incubated with the secondary antibodies for 2 hours at room temperature. The membranes were visualized using enhanced chemiluminescence solution (Pierce Biotechnology, Inc. USA). The experimental results were analyzed by ImageJ analysis software. The following antibodies were recorded: anti-GAPDH, anti-Flagtag, anti-HIF1α, anti-YTHDF2 (Proteintech, Wuhan, Hubei, China), anti-AKT, anti-Phospho-AKT (Ser473), anti-ERK1/2, anti-Phospho-ERK1/2 (Thr202/Tyr204, Cell Signaling Technologies, Danvers, MA, USA), anti-Phospho-mTOR (Ser2448), anti-mTOR, anti-E-cadherin, anti-N-cadherin, anti-Vimentin, anti-Snail1, anti-METTL3, anti-METTL14.
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