4.1 Reagents Required for Blocking

Reagents Required for Blocking
1. Proteins Used in Blocking Buffers - Comparison of Proteins for Blocking Buffers in Western Blotting
Protein Buffer Recommended Concentration Suitable Membranes
BSA TBST, TBS 0.2%—5% (W/V) NC, PVDF
Non-fat milk powder TBST, TBS 3%—5% (W/V) NC, PVDF
Amersham ECL Prime Blocking Buffer (contains non-fat milk) TBST, TBS 2%—5% (W/V) NC, PVDF
Casein TBST, TBS 5% (W/V) NC, PVDF
Fish gelatin TBST, TBS 2%—10% (W/V) NC, PVDF
2. Proteins Used in Blocking Buffers - Blocking Conditions for Different Proteins
Protein Blocking Conditions
Non-fat milk powder The most commonly used blocking buffer, typically prepared by adding 1-5% non-fat milk powder to TBST or TBS solution. Non-fat milk powder is inexpensive and provides a clean background. However, it should be noted that 5% non-fat milk powder solution is prone to spoilage and is not suitable for reuse.
Fish gelatin Fish gelatin contains fewer hydrogen-bonding amino groups compared to mammalian-derived gelatin, thus often resulting in lower background. It is usually used at a concentration of 2% and dissolves easily below 40°C. However, it contains some competitive reactants such as biotin.
BSA BSA is relatively inexpensive and suitable for most experiments. The concentration in TBST or TBS is reduced to 0.3%-3%. In cases of high background, 0.1% Tween-20 can be optionally added. For phosphorylation experiments, it is recommended to use BSA at a concentration of 2%.
Serum 10% horse or fetal bovine serum is often used, with the solution containing 0.02% sodium azide. This type of blocking buffer is expensive and may cause cross-reactivity.
Notes:
The content and quality of non-fat milk powder may vary between different manufacturers and batches.
Due to the specificity of each antigen-antibody interaction, there is no single optimal blocking buffer.
Determining the optimal blocking buffer and concentration is a key step in successful immunoassays. Appropriate conditions can improve the signal-to-noise ratio of imaging, i.e., image quality.
A low concentration of blocking buffer may lead to high background (darkening of the membrane).
On the other hand, excessive blocking buffer can affect the binding of antibodies to antigens.
3. Blocking Buffer Solutions
TBST and TBS are commonly used as buffers for blocking solutions. For chemiluminescence detection, TBST buffer is recommended. For fluorescence detection, TBS buffer is recommended.