Western Blot Protocol
Materials:
Sample Preparation:
Protein samples (cell lysates or purified proteins)
Lysis buffer (RIPA or other suitable buffer)
Protease and phosphatase inhibitors
BCA or Bradford protein assay kit
SDS-PAGE:
SDS-PAGE gel (10% or 12% depending on the protein size)
Running buffer (1X Tris-Glycine-SDS)
Sample loading buffer (4X Laemmli buffer)
Protein ladder
Transfer:
PVDF or nitrocellulose membrane
Transfer buffer (1X Tris-Glycine, 20% methanol)
Filter papers and sponges
Transfer apparatus (wet or semi-dry)
Blocking and Antibody Incubation:
Blocking buffer (5% non-fat dry milk or BSA in TBST)
Primary antibody (specific to the target protein)
Secondary antibody (HRP-conjugated anti-mouse, anti-rabbit, etc.)
Detection:
ECL detection reagent
X-ray film or imaging system
Additional:
Pipettes and tips
Incubator or rocking platform
Gel documentation system
Procedure:
1. Sample Preparation:
Harvest cells or tissue samples and wash with cold PBS.
Lyse the cells using an appropriate lysis buffer containing protease and phosphatase inhibitors.
Centrifuge at 12,000 x g for 15 minutes at 4°C to remove cell debris.
Collect the supernatant and determine protein concentration using BCA or Bradford assay.
2. SDS-PAGE:
Mix protein samples with 4X sample loading buffer (final concentration 1X) and heat at 95°C for 5 minutes.
Load equal amounts of protein (usually 20-30 µg) into each well of the SDS-PAGE gel.
Load the protein ladder into a separate well.
Run the gel at 80-100V through the stacking gel and then at 120-150V through the resolving gel until the dye front reaches the bottom of the gel.
3. Transfer:
Pre-soak the PVDF membrane in methanol for 30 seconds, then equilibrate in transfer buffer.
Assemble the transfer sandwich: sponge, filter paper, gel, PVDF membrane, filter paper, sponge.
Place the sandwich into the transfer cassette and immerse in transfer buffer.
Transfer at 100V for 60-90 minutes (wet transfer) or 25V for 7-10 minutes (semi-dry transfer).
4. Blocking:
After transfer, block the membrane in 5% non-fat dry milk or 5% BSA in TBST for 1 hour at room temperature with gentle shaking.
5. Primary Antibody Incubation:
Dilute the primary antibody in blocking buffer according to the manufacturer’s recommendation.
Incubate the membrane with the primary antibody overnight at 4°C with gentle shaking.
6. Washing:
Wash the membrane 3 times for 5 minutes each with TBST.
7. Secondary Antibody Incubation:
Dilute the HRP-conjugated secondary antibody in blocking buffer.
Incubate the membrane for 1 hour at room temperature with gentle shaking.
8. Washing:
Wash the membrane 3 times for 5 minutes each with TBST.
9. Detection:
Prepare ECL detection reagent according to the manufacturer’s instructions.
Apply ECL reagent evenly over the membrane and incubate for 1-2 minutes.
Detect the signal using an imaging system or by exposing the membrane to X-ray film.
10. Analysis:
Quantify the bands using densitometry software if required.
Normalize the target protein band intensity to the loading control (e.g., GAPDH, β-actin).
Tips:
Always include a loading control to verify equal protein loading.
Optimize antibody concentrations and incubation times to minimize background.
Use fresh reagents and ensure all equipment is clean to avoid contamination.