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Total protein is extracted from cells or tissues, followed by lysis, centrifugation, purification, protein quantification and thermal denaturation to prepare standardized loading samples, which serve as the fundamental pre-process for Western blot (WB) experiments.

Separation gel and stacking gel with appropriate concentrations are prepared according to the molecular weight of the target proteins. After complete gel polymerization, the gel is applied to electrophoretically separate protein components of different molecular weights.

Load protein samples and protein molecular weight markers into gel wells, conduct electrophoresis under optimized voltage, and separate distinct protein fractions based on differences in molecular weight.

Layer filter paper, polyacrylamide gel, transfer membrane, and another piece of filter paper in sequence, fully eliminate air bubbles between layers, assemble the transfer cassette, and place it into the transfer tank filled with transfer buffer.

Apply an electric current to transfer proteins from the gel to NC/PVDF membrane and immobilize protein bands.

After protein transfer, verify the transfer efficiency first. Subsequently, incubate the membrane with blocking buffer to cover blank membrane areas and block non-specific antibody binding sites.

Incubate the membrane with primary antibody followed by secondary antibody sequentially. Thoroughly wash the membrane between incubations to remove unbound free antibodies.

If multiple target proteins need to be detected on the same membrane, strip the bound original antibodies with stripping buffer, re-block the membrane, and incubate with new primary and secondary antibodies.

Add a chemiluminescent substrate for exposure and imaging, capture images of protein bands, quantify band signal intensity via gray-value analysis, and compare the relative target protein expression levels among different groups.