What Are the Steps of Western Blotting?
Steps of Western Blotting
Your comprehensive guide to protein analysis with Affigen Lab Insights
1. Sample Preparation
Proper preparation of protein samples is crucial for successful Western Blotting. Ensure that proteins are denatured and reduced appropriately before loading.
- Lyse cells or tissue to extract proteins.
- Quantify protein concentration.
- Add loading buffer with SDS and reducing agent.
- Boil samples for denaturation.
2. Gel Electrophoresis
Separate proteins based on molecular weight using SDS-PAGE.
- Prepare polyacrylamide gel.
- Load protein samples and molecular weight markers.
- Run the gel at appropriate voltage until separation is achieved.
3. Protein Transfer
Transfer proteins from the gel onto a PVDF or nitrocellulose membrane for detection.
- Equilibrate gel and membrane in transfer buffer.
- Assemble transfer sandwich.
- Run transfer using wet or semi-dry method.
4. Blocking
Block nonspecific binding sites on the membrane to reduce background signal.
- Incubate membrane in 5% non-fat milk or BSA in TBST buffer.
- Typically 1 hour at room temperature or overnight at 4°C.
5. Antibody Incubation
Detect target proteins with specific primary and secondary antibodies.
- Incubate with primary antibody specific to target protein.
- Wash membrane to remove unbound antibody.
- Incubate with enzyme-conjugated secondary antibody.
6. Detection
Visualize the protein bands using chemiluminescent or fluorescent substrates.
- Add substrate compatible with the enzyme on the secondary antibody.
- Capture image using a gel documentation system.
- Analyze band intensity for protein quantification.
