Western Blot Hub

Best Blocking Buffer for Western Blot: Complete Guide

Choosing the right blocking buffer is essential for successful western blotting. The blocking buffer prevents non-specific antibody binding and affects signal quality. This comprehensive guide compares different blocking agents, provides recipes, and offers optimization tips to help you select the best blocking buffer for your specific application.

Overview

Blocking buffers prevent non-specific binding of antibodies to the membrane. Common blocking agents include:

  • Milk: Non-fat dry milk, cost-effective, widely used
  • BSA: Bovine serum albumin, required for phosphoproteins
  • Casein: Purified casein, alternative blocking agent
  • Other: Fish gelatin, serum, commercial blocking solutions

The choice depends on your protein type, detection method, and budget.

Milk Blocking Buffer

Recipe

5% Milk in TBST:

  • 5 g non-fat dry milk
  • 100 mL TBST (Tris-buffered saline with 0.1% Tween-20)
  • Mix well, filter if needed
  • Store at 4°C, use within 1 week
  • Advantages: Cost-effective, widely used, good blocking
  • Disadvantages: Contains phosphatases (not for phosphoproteins)
  • Use for: General protein detection, non-phosphorylated proteins

BSA Blocking Buffer

Recipe

5% BSA in TBST:

  • 5 g BSA (bovine serum albumin)
  • 100 mL TBST (Tris-buffered saline with 0.1% Tween-20)
  • Mix well, filter if needed
  • Store at 4°C, use within 1 week
  • Advantages: No phosphatase activity, required for phosphoproteins, consistent
  • Disadvantages: More expensive than milk
  • Use for: Phosphorylated proteins, when milk gives high background

Casein Blocking Buffer

Recipe

Casein in TBST:

  • 1-2% casein in TBST
  • Mix well, may need heating to dissolve
  • Filter before use
  • Advantages: Good blocking, alternative to milk
  • Disadvantages: May be more expensive, less commonly used
  • Use for: Alternative blocking agent

Selection Guide

Choose Milk When:

  • Detecting non-phosphorylated proteins
  • Cost is a concern
  • Routine western blotting
  • General protein detection

Choose BSA When:

  • Detecting phosphorylated proteins (required)
  • Milk gives high background
  • Need consistent, reproducible results
  • Budget allows for higher cost

Complete Recipes

TBST (Base Buffer)

  • 20 mM Tris-HCl, pH 7.5
  • 150 mM NaCl
  • 0.1% Tween-20
  • Adjust pH to 7.5

Blocking Conditions

  • Block for 1-2 hours at room temperature
  • Or block overnight at 4°C
  • Use 5% blocking agent in TBST
  • Ensure complete membrane coverage

Related Articles

Blocking Protocol

Complete blocking guide

Milk vs BSA

Compare blocking agents