Western Blot Protocol

Materials:

  1. 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

  2. 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

  3. Transfer:

    • PVDF or nitrocellulose membrane

    • Transfer buffer (1X Tris-Glycine, 20% methanol)

    • Filter papers and sponges

    • Transfer apparatus (wet or semi-dry)

  4. 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.)

  5. Detection:

    • ECL detection reagent

    • X-ray film or imaging system

  6. Additional:

    • Pipettes and tips

    • Incubator or rocking platform

    • Gel documentation system

Procedure:

1. Sample Preparation:
  1. Harvest cells or tissue samples and wash with cold PBS.

  2. Lyse the cells using an appropriate lysis buffer containing protease and phosphatase inhibitors.

  3. Centrifuge at 12,000 x g for 15 minutes at 4°C to remove cell debris.

  4. Collect the supernatant and determine protein concentration using BCA or Bradford assay.

2. SDS-PAGE:
  1. Mix protein samples with 4X sample loading buffer (final concentration 1X) and heat at 95°C for 5 minutes.

  2. Load equal amounts of protein (usually 20-30 µg) into each well of the SDS-PAGE gel.

  3. Load the protein ladder into a separate well.

  4. 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:
  1. Pre-soak the PVDF membrane in methanol for 30 seconds, then equilibrate in transfer buffer.

  2. Assemble the transfer sandwich: sponge, filter paper, gel, PVDF membrane, filter paper, sponge.

  3. Place the sandwich into the transfer cassette and immerse in transfer buffer.

  4. Transfer at 100V for 60-90 minutes (wet transfer) or 25V for 7-10 minutes (semi-dry transfer).

4. Blocking:
  1. 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:
  1. Dilute the primary antibody in blocking buffer according to the manufacturer’s recommendation.

  2. Incubate the membrane with the primary antibody overnight at 4°C with gentle shaking.

6. Washing:
  1. Wash the membrane 3 times for 5 minutes each with TBST.

7. Secondary Antibody Incubation:
  1. Dilute the HRP-conjugated secondary antibody in blocking buffer.

  2. Incubate the membrane for 1 hour at room temperature with gentle shaking.

8. Washing:
  1. Wash the membrane 3 times for 5 minutes each with TBST.

9. Detection:
  1. Prepare ECL detection reagent according to the manufacturer’s instructions.

  2. Apply ECL reagent evenly over the membrane and incubate for 1-2 minutes.

  3. Detect the signal using an imaging system or by exposing the membrane to X-ray film.

10. Analysis:
  1. Quantify the bands using densitometry software if required.

  2. 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.