How to quantitatively determine the expression of a certain protein in cells using Western blot?
Western blot is generally used for qualitative analysis of protein presence and to study their expression changes under different conditions. The basic steps are as follows:
1. Sample Preparation:
Lyse cells using an appropriate lysis buffer and determine the total protein concentration using protein concentration measurement methods such as BCA or Bradford assay.
2. SDS-PAGE:
Select an appropriate gel based on the expected size of the protein and load equal amounts of total protein into each lane.
3. Transfer:
Transfer proteins from the SDS-PAGE to a PVDF or nitrocellulose membrane.
4. Blocking:
Use non-specific proteins (such as non-fat milk or BSA) to block unbound sites on the membrane.
5. Primary Antibody Incubation:
Incubate with specific antibodies against the target protein.
6. Secondary Antibody Incubation:
Incubate with secondary antibodies against the primary antibody, which are usually labeled with a fluorescent substance or enzyme.
7. Detection:
Detect the signal of the target protein using chemiluminescent reagents or other methods.
8. Quantification:
Analyze the gray values of the bands using image analysis software, such as ImageJ. To correct for loading differences, internal control proteins such as GAPDH or β-actin are often used for normalization. Calculate the relative expression level of the target protein based on the obtained gray values and those of the internal control.
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