Step 1: Sample Preparation

Proper sample preparation is crucial for successful western blotting. The method depends on your sample type:

• Cell lysates: Lyse cells in appropriate lysis buffer with protease and phosphatase inhibitors
• Tissue samples: Homogenize tissue in lysis buffer using mechanical methods
• Purified proteins: Dilute to appropriate concentration in sample buffer

Determine protein concentration using BCA, Bradford, or Lowry assay. Mix samples with Laemmli sample buffer (typically 1:1 ratio) and heat at 95°C for 5 minutes to denature proteins. This step is essential for proper protein separation during electrophoresis.

Step 2: SDS-PAGE Electrophoresis

Load equal amounts of protein (typically 20-50 μg) into each well of the SDS-PAGE gel. The exact amount depends on your target protein abundance and detection method. Include molecular weight markers in at least one lane for size reference.

Run electrophoresis at constant voltage (typically 80-120V) until the dye front reaches the bottom of the gel. The running time depends on gel percentage and protein size. Monitor the run to ensure proteins don't run off the gel.

After electrophoresis, the gel can be stained with Coomassie Blue to verify protein separation, or proceed directly to transfer.

Step 3: Protein Transfer

Transfer proteins from gel to membrane using either wet transfer or semi-dry transfer methods:

• Wet transfer: Use 100V for 1 hour or 30V overnight at 4°C. This method is more reliable for large proteins (>100 kDa)
• Semi-dry transfer: Use 15V for 30-60 minutes. Faster but may be less efficient for large proteins

After transfer, verify successful transfer by staining the membrane with Ponceau S or by checking the gel for remaining proteins.

Step 4: Membrane Blocking

Block the membrane with 5% non-fat milk or 3-5% BSA in TBST for 1 hour at room temperature with gentle shaking. Blocking prevents non-specific antibody binding and reduces background signal.

The choice between milk and BSA depends on your antibody. Some antibodies work better with BSA, especially when detecting phosphorylated proteins, as milk contains casein which can interfere with phospho-specific antibodies.

Step 5: Primary Antibody Incubation

Dilute primary antibody in blocking solution according to manufacturer's recommendations (typically 1:1000 to 1:5000). Incubate membrane with primary antibody overnight at 4°C or for 1-2 hours at room temperature with gentle shaking.

Optimal antibody concentration should be determined empirically. Too high concentration can increase background, while too low may result in weak or no signal.

Step 6: Washing

Wash membrane 3-5 times with TBST, 5 minutes each wash, to remove unbound primary antibody. Thorough washing is essential to reduce background signal. Increase washing time or number of washes if background is high.

Step 7: Secondary Antibody Incubation

Dilute HRP-conjugated secondary antibody in blocking solution (typically 1:5000 to 1:10000). Incubate membrane for 1 hour at room temperature with gentle shaking.

Ensure the secondary antibody is specific to the species and isotype of your primary antibody. For example, if using a mouse IgG primary antibody, use an anti-mouse IgG secondary antibody.

Step 8: Final Washing

Wash membrane 3-5 times with TBST, 5 minutes each wash, to remove unbound secondary antibody. This final washing step is critical for clean results with minimal background.

Step 9: Detection

For chemiluminescent detection, incubate membrane with ECL substrate for 1-5 minutes, then expose to X-ray film or capture with imaging system. Exposure time varies depending on signal strength (typically 1 second to 10 minutes).

For fluorescent detection, scan membrane with appropriate excitation wavelength using a fluorescence imaging system. Protect membranes from light during fluorescent detection to prevent photobleaching.