Western Blotting Protocol for Protein Analysis

Western blotting was performed as described before (Xue et al., 2017 (link)). Each lane was loaded 20–40 μg protein, and the PVDF membrane was blocked with 5% skim milk in 0.1% TBST for 1 h. After washing the membrane for three times, the membrane was incubated with primary antibody at 4°C overnight. HRP-conjugated secondary antibody and ECL kit were used to detect primary antibody with Bio-Rad Exposure System. The gray value of each lane was calculated with ImageJ.

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Hang W., He B., Chen J., Xia L., Wen B., Liang T., Wang X., Zhang Q., Wu Y., Chen Q, & Chen J. (2018). Berberine Ameliorates High Glucose-Induced Cardiomyocyte Injury via AMPK Signaling Activation to Stimulate Mitochondrial Biogenesis and Restore Autophagic Flux. Frontiers in Pharmacology, 9, 1121.

Publication 2018

Exposure System

Antibody Membrane Milk Protein Pvdf

Corresponding Organization : Hubei University of Chinese Medicine

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Variable analysis

independent variables

Protein loading amount (20-40 μg)

dependent variables

Gray value of each lane (measured with ImageJ)

control variables

PVDF membrane blocking with 5% skim milk in 0.1% TBST for 1 h
Primary antibody incubation at 4°C overnight
HRP-conjugated secondary antibody and ECL kit used for detection
Bio-Rad Exposure System used for detection

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