Total protein extract from equal numbers of cells was prepared using the TCA extraction method. An equal amount of proteins was resolved in an 8% SDS–PAGE gel and transferred to an Immobilon-FL (Millipore, Darmstadt, Germany) membrane using a wet electrotransfer system (Bio-Rad, Munich, Germany). The membranes were incubated with an anti-HA (Abcam, Cambridge, UK) antibody followed by anti-rabbit immunoglobulin G antibody coupled with Alexa Fluor 680 (Molecular Probes, Life Technologies, Darmstadt, Germany) and scanned on the Odyssey Clx imager (Li-Cor, Bad Homburg, Germany). The Western blots were quantified using AIDA software (Raytek, Berlin, Germany).
Wet electrotransfer system
Manufactured by Bio-Rad
Sourced in Germany, China
The Wet electrotransfer system is a laboratory equipment used for the transfer of proteins from a polyacrylamide gel to a membrane or other solid support for subsequent detection and analysis. The system allows for the efficient and consistent transfer of proteins from the gel to the membrane, which is a crucial step in various biochemical and molecular biology techniques.
Automatically generated - may contain errors
2 protocols using wet electrotransfer system
1
Western Blot Protein Quantification
2
MFGM Treatment Protein Extraction
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C 2 C 12 cells were treated with 200 μg/mL of either MFGM or MFGM P2 for 48 h, washed twice with PBS buffer and homogenized in lysis buffer. Total protein was extracted and harvested by scraping with a modified radioimmunoprecipitation assay buffer containing 100 nM phenylmethylsulfonyl fluoride for 30 min [26] . Following centrifugation at 10000 rpm for 15 min at 4℃, the supernatant was sonicated. The protein concentration was quantified using a BCA kit (Solarbio, China). Proteins (100 μg) were loaded onto a 1-D SDS gel (10 % polyacrylamide). Next, proteins were transferred onto a nitrocellulose filter membrane (PPLYGEN, China) using a wet electrotransfer system (Bio-Rad, USA)for 4 h at 200 mA. The membranes were blocked with 5 % non-fat dry milk in Tris-buffered saline with Tween-20 (TBST)
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