Protein lysate preparation and Western blot analysis

YL Yung-Wei Lin
YW Yu-Ching Wen
CC Chih-Ying Chu
MT Min-Che Tung
YY Yi-Chieh Yang
KH Kuo-Tai Hua
KP Ke-Fan Pan
MH Michael Hsiao
WL Wei-Jiunn Lee
MC Ming-Hsien Chien
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Protein lysates were prepared as described previously23. Total cellular proteins were determined using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). Equal amounts of protein were subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) or SDS-PAGE gradient gels (BIOTOOLS, New Taipei City, Taiwan) and then electrophoretically transferred to PVDF membranes (Bio-Rad). Membranes were then incubated with indicated primary antibodies and horseradish peroxidase-conjugated secondary antibodies. After washing, blots were incubated with the ECL Western blotting reagent, and chemiluminescence was detected by the chemiluminescence imaging system, MultiGel-21 (TOP BIO, New Taipei City, Taiwan). Image-Pro Plus software (Media Cybernetics, Silver Spring, MD) was used to quantify the density of specific bands.

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