Western Blot Blocking: Step-by-Step Protocol

Blocking is a critical step in western blotting that prevents non-specific binding of antibodies to the membrane. This detailed protocol covers blocking solution preparation, incubation conditions, and optimization techniques to achieve low background and high signal-to-noise ratio.

Overview

Blocking prevents non-specific binding of antibodies to the membrane by saturating unoccupied protein-binding sites. This step is essential for:

Reducing background signal
Improving signal-to-noise ratio
Preventing false positive results
Ensuring specific antibody binding

The choice of blocking solution (milk vs BSA) and blocking conditions significantly affect the final results. This protocol provides detailed guidance for optimal blocking.

Blocking Solutions

Two main blocking solutions are commonly used, each with specific advantages:

5% Non-Fat Milk in TBST

Standard blocking solution

Preparation:

Dissolve 5 g non-fat dry milk powder in 100 mL TBST
Mix well until completely dissolved (5-10 minutes)
Filter through cheesecloth if particles remain
Store at 4°C for up to 1 week

Applications:

Most general applications
Cost-effective
Works well for most antibodies

Limitations: Contains casein which can interfere with phospho-specific antibodies

3-5% BSA in TBST

For phosphorylated proteins

Preparation:

Dissolve 3-5 g BSA (Fraction V) in 100 mL TBST
Mix gently to avoid foaming
Filter through 0.45 μm filter if necessary
Store at 4°C, use within 1 week

Applications:

Phosphorylated proteins
When milk causes high background
Some sensitive antibodies

Advantages: No casein interference, lower background for phospho-antibodies

Important Selection Guide

For phosphorylated proteins: Always use BSA, not milk
For most other proteins: Milk works well and is cost-effective
If high background with milk: Try BSA instead
Test both: Some antibodies work better with one or the other

Step-by-Step Procedure

Step 1: Prepare Blocking Solution

Prepare blocking solution fresh or use stored solution (check expiration). For best results, prepare fresh:

For 5% Milk:

Dissolve 5 g non-fat dry milk powder in 100 mL TBST
Mix well by swirling or gentle inversion (5-10 minutes)
Filter through cheesecloth or 0.45 μm filter if particles remain
Store at 4°C if not using immediately

For 3-5% BSA:

Dissolve 3-5 g BSA in 100 mL TBST
Mix gently to avoid foaming
Filter through 0.45 μm filter if necessary
Store at 4°C

Step 2: Remove Ponceau S Stain (Optional)

If you verified transfer with Ponceau S staining, wash membrane briefly with TBST:

2-3 quick rinses, 30 seconds each
This step is optional - Ponceau S will be removed during subsequent washes
Drain excess TBST but do not let membrane dry

Step 3: Transfer Membrane to Blocking Solution

Place membrane protein-side up in blocking solution. Use adequate volume:

Mini-gel (8 x 10 cm): 10-20 mL
Larger membranes: 30-50 mL
Ensure membrane floats freely and is completely submerged
Remove any air bubbles by gently tapping or using forceps

Critical: Membrane must be completely covered. Insufficient coverage causes high background at uncovered areas.

Step 4: Incubate with Gentle Agitation

Place container on a rocker or shaker set to gentle speed (20-30 rpm):

Standard: 1 hour at room temperature (20-25°C)
For high background: Extend to 2 hours or use 10% milk
Alternative: Overnight at 4°C (12-16 hours) - may improve efficiency

Too vigorous shaking can damage the membrane or cause uneven blocking. Use gentle, consistent agitation.

Step 5: Proceed to Primary Antibody

After blocking:

Do not wash the membrane
Drain excess blocking solution by touching edge to paper towel
Do not let membrane dry
Immediately proceed to primary antibody incubation
Use the same type of blocking solution (milk or BSA) for diluting primary antibody

Optimization Tips

General Guidelines

Ensure membrane is completely submerged - insufficient coverage causes high background
Use gentle shaking (20-30 rpm) - too vigorous can damage membrane
Blocking can be done at 4°C overnight if convenient
Prepare blocking solution fresh when possible
Use adequate volume - membrane should float freely
Do not reuse blocking solution

For Phosphorylated Proteins

Always use BSA, not milk - milk contains casein which interferes with phospho-specific antibodies
Use 3-5% BSA in TBST
May need to extend blocking time to 2 hours

For High Background Issues

Extend blocking time to 2 hours
Increase blocking concentration to 10% milk
Try BSA instead of milk
Ensure membrane was properly blocked (completely submerged)

Troubleshooting

High Background

Solutions:

Extend blocking time to 2 hours
Increase blocking concentration to 10% milk
Try BSA instead of milk
Ensure membrane was completely submerged during blocking
Check that blocking solution was fresh

Uneven Blocking