Antibody dilution buffer is used to dilute primary and secondary antibodies as well as some detecting reagents.
Primary Antibody Dilution Buffer
- 1%BSA (blocking & stabilizer)
- 0.1% cold fish skin gelatin (blocking)
- 0.5% Triton X-100 (penetration enhancer)
- 0.05% sodium azide (preservative)
- 0.01M PBS, pH 7.2-7.4
- Mix well and store at 4 ºC.
Note: Do not use it to dilute HRP conjugated antibody as the sodium azide is inhibitor of HRP. Do not use it to dilute Alkaline Phosphatase (AP) conjugated antibody as the phosphate in PBS is an inhibitor of AP.
HRP-conjugated Primary Antibody Dilution Buffer:
- 1% BSA (stabilizer)
- 0.1% cold fish skin gelatin (blocking)
- 0.05% Thimerosal (preservative)
- 0.01M PBS, pH 7.2
To make 100 ml of the buffer, add 1 g BSA, 0.1 g fish skin gelatin, 0.05 g thimerosal in 100 ml 0.01M PBS, pH 7.2
Note: Antibodies diluted using this buffer can be stored at 4 ºC for 6 months without reducing binding activity.
Secondary Antibody Dilution Buffer:
- 0.01M PBS, pH 7.2
- 0.05% Tween 20
To make 1000 ml of the buffer, add 0.5 ml of Tween 20 in 1000 ml, 0.01M PBS, pH 7.2
Note: 1) Antibodies diluted using this buffer can be stored at 4 ºC for 6 months without reducing binding activity; 2) Do not use BSA or other serum containing reagents to dilute secondary antibodies since they may bind to BSA or serum therefore reducing antibody affinity; 3) Using TBS to dilute secondary antibodies often produces weaker staining. So use TBS only for the antibodies with high background staining or for alkaline phosphatase conjugated antibodies since phosphate is a inhibitor of alkaline phosphatase; 4) This buffer can also be used for diluting HRP conjugated antibodies since there is no sodium azide in buffer.
AP-Streptavidin Dilution Buffer:
- 0.05M TBS, pH 7.6
- 0.05% Tween 20
To make 1000 ml of the buffer, add 0.5 ml of Tween 20 in 1000 ml, 0.05M TBS, pH 7.6
Note: 1) Antibodies diluted using this buffer can be stored at 4 ºC for 6 months without reducing binding activity; 2) PBS can not be used to dilute Streptavidin-AP since phosphate inhibits alkaline phosphatase activity.
Fluorescent Dye (Avidin-FITC, Avidin-Texas Red, Avidin-AMCA) Dilution Buffer:
- 0.01M PBS, pH 7.2
- 0.05% Tween 20
To make 1000 ml of the buffer, add 0.5 ml of Tween 20 in 1000 ml, 0.01M PBS, pH 7.2
Note: 1)Antibodies diluted using this buffer can be stored at 4 ºC for 6 months without reducing binding activity; 2) Do not use serum containing solution to dilute avidin/streptavidin conjugates since serum may contain biotin therefore reducing reaction activity for avidin/streptavidin conjugates.