Antibody Dilution Buffers

Tony Rook

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Nov 16, 2015
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Please find the following media preparation methods for:

Antibody Dilution Buffers



Primary Antibody Dilution Buffer:

1%BSA (stabilizer and blocking)

0.1% cold fish skin gelatin (blocking)

0.05% sodium azide (preservative)

0.01M PBS pH7.2 (TBS pH7.6 used in primary antibody
dilution buffer produces weaker staining)

Note:
1) Antibodies diluted using this buffer can be stored at 4 C for 6 months without reducing binding activity.
2) This buffer can not be used for diluting HRP conjugated antibodies since sodium azide is an inhibitor of HRP.




HRP-conjugated Primary Antibody Dilution Buffer:

1%BSA (stabilizer)

0.1% cold fish skin gelatin (blocking)

0.05% Thimerosal (preservative)

0.01M PBS pH7.2

Note:
Antibodies diluted using this buffer can be stored at 4 C for 6 months without reducing binding activity.




Secondary Antibody Dilution Buffer:

0.01M PBS, pH 7.2

0.05% sodium azide (preservative)

Note:
1) Antibodies diluted using this buffer can be stored at 4 C 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.




HRP-Streptavidin Dilution Buffer:

0.01M PBS, pH 7.2

0.05% thimerosal (preservative)

Note:
Antibodies diluted using this buffer can be stored at 4 C for 6 months without reducing binding activity.




AP-Streptavidin Dilution Buffer:

0.05M TBS, pH 7.6

0.05% thimerosal (preservative)

Note:
1) Antibodies diluted using this buffer can be stored at 4 C for 6 months without reducing binding activity.
2) PBS can not be used to dilute Streptavidin-AP since phosphate inhibit alkaline phosphatase activity.




Fluorescent Dye (Avidin-FITC, Avidin-Texas Red, Avidin-AMCA) Dilution Buffer:

0.01M PBS, pH 7.2

0.05% thimerosal (preservative)

Note:
1) Antibodies diluted using this buffer can be stored at 4C 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.



Link Reference:
http://www.ihcworld.com/_protocols/antibody_diluents/ab_dilution_buffer.htm