Blocking

In Western blotting, blocking prevents nonspecific binding of antibodies to the membrane (PVDF or nitrocellulose). The most common blocking agents are protein-based and occupy free binding sites on the membrane still present after protein transfer.

Non-fat (skimmed) dry Milk

Concentration: 3–5 % (w/v) in TBST or PBST

Advantages

• Inexpensive
• Works well for most antibodies
• Good general-purpose blocker
• Fast (30–60 min at RT or overnight at 4°C)

Disadvantages

• Contains casein (a phosphoprotein) → not ideal for phospho-specific antibodies
• Can interfere with biotin/avidin systems
• Batch to batch variability
• Different skimmed milk preparations from different suppliers may have different blocking properties

BSA (Bovine Serum Albumin)

Concentration: 3–5 % (w/v) in TBST or PBST

Advantages

• Better for phospho-protein detection
• More consistent reagent compared to milk
• Minimal batch to batch variability

Disadvantages

• More expensive than milk
• Sometimes less efficient blocking

Commercial Blocking Buffers

Examples: SuperBlock™, StartingBlock™, Odyssey® Blocking Buffer, Intercept® (LI-COR)

Advantages

• Optimized formulations
• Low background
• Good for fluorescent Western blots
• Minimal batch to batch variability

Disadvantages

• Expensive
• Not always better than BSA or milk