How to Quantify Protein Expression in Cells Using Western Blot?
Western blot (protein blotting) is typically used for qualitative analysis of protein presence and studying expression changes under different conditions. The general steps are as follows:
Sample Preparation
Use appropriate lysis buffer to lyse the cells, and measure the total protein concentration using a protein assay method (such as BCA or Bradford).
SDS-PAGE
Choose the appropriate gel based on the expected protein size and load the same amount of total protein into each lane.
Transfer
Transfer the proteins from the SDS-PAGE gel to a PVDF or nitrocellulose membrane.
Blocking
Block unbound sites on the membrane using non-specific proteins (e.g., skim milk or BSA).
Primary Antibody Incubation
Incubate the membrane with a specific antibody against the target protein.
Secondary Antibody Incubation
Incubate with a secondary antibody against the primary antibody, which is typically labeled with a fluorescent dye or enzyme.
Detection
Detect the target protein signal using chemiluminescent reagents or other methods.
Quantification
Analyze the band intensity using imaging analysis software like ImageJ. To correct for loading differences, normalize to a housekeeping protein like GAPDH or β-actin. Calculate the relative expression level of the target protein based on the intensity values of the bands and the housekeeping protein.
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