Western Blot Protocol

1. Cell Lysis

• Wash adherent cells 2× with ice-cold PBS; suspend cells in pre-chilled lysis buffer (e.g., RIPA with inhibitors).
• Incubate on ice for 15-30 min with occasional vortexing.
• Clarify lysate by centrifugation at 12,000-20,000 × g, 4 °C, 10-15 min.
• Collect supernatant; avoid disturbing the pellet.

2. Tissue Homogenization

• Snap-freeze tissue in liquid nitrogen; grind to powder.
• Homogenize in lysis buffer using a mechanical homogenizer or sonicator (pulse on ice).
• Clarify as above.

3. Protein Quantification

• Perform BCA assay according to manufacturer instructions.
• Prepare equal protein amounts (typically 20-40 μg per lane) in a final volume of 15-25 μL.

4. Denaturation

• Mix protein samples with 4×-6× SDS loading buffer containing reducing agent.
• Heat at 95 °C for 5-10 min (cytosolic and nuclear proteins). For membrane proteins, use 37 °C for 30 min to reduce aggregation.
• Briefly spin to collect condensation; load immediately or store at −20 °C.

1. Gel Selection

• 8% gel: proteins >100 kDa
• 10-12% gel: proteins 20-100 kDa
• 15% gel: proteins <20 kDa (e.g., histones)

2. Assembly and Loading

• Assemble gel in electrophoresis tank; fill with 1× running buffer.
• Load protein samples and prestained molecular weight marker.
• Run at 80 V through the stacking gel (approx. 30 min), then increase to 120-200 V through the resolving gel until the dye front reaches the bottom.

1. Membrane Preparation

• PVDF: Immerse in 100% methanol for 30 s, then equilibrate in transfer buffer.
• Nitrocellulose: Equilibrate directly in transfer buffer.

2. Transfer Setup

• Assemble transfer sandwich: anode → filter paper → membrane → gel → filter paper → cathode.
• Roll out air bubbles meticulously using a glass rod or pipette; trapped bubbles cause patchy transfer.

3. Transfer Conditions

4. Transfer Validation

• Stain membrane with Ponceau S (reversible) or stain the gel post-transfer with Coomassie to confirm complete transfer.

1. Primary Antibody

• Dilution: 1:500-1:5,000 (typically 1:1,000; titrate based on signal intensity)
• Diluent: Blocking buffer or commercial antibody diluent
• Incubation: Overnight at 4 °C with gentle agitation for optimal specificity

2. Washing

• Wash membrane 3 × 5-10 min with TBST at room temperature.

3. Secondary Antibody

• Dilution: 1:5,000-1:10,000
• Diluent: Blocking buffer or TBST
• Incubation: 1 h at room temperature with gentle rocking

4. Final Washing

• Wash membrane 3 × 5-10 min with TBST to remove unbound secondary antibody.

1. Chemiluminescence (ECL)

• Mix ECL substrate (peroxide and luminol solutions) 1:1.
• Incubate membrane for 1-5 min; drain excess reagent.
• Capture image using chemiluminescence imager or X-ray film.
• Perform multiple exposures (short to long) to avoid signal saturation.

2. Fluorescence Detection

• Use IRDye-conjugated secondary antibodies.
• Image on a fluorescence scanner.