Does anybody know how to choose a proper diluent for my primary antibody? I have a homemade antibody that I am optimising and do not know which diluent to choose between TBS, TBST, PBS?? also I read some add blocking buffer to the diluent?
Hi Rawda Elshennawy
Most protocols recommend a particular diluent depending on the antibody you are using. I would first check the manufactuers recommendation. If this is not available, I would use the blocking buffer. This is typically either TBST/PBST with 3-5% of protein such as BSA or serum.
Good luck!
TBST/PBST can be stored at room temperature. The proteins will have to be aliquoted and frozen if you are planning on keeping them for a while. Simply thaw and add 3-5% of BSA/serum prior to your antibody incubation!
It is essential that you calibrate the ionic strength and pH of the antibody diluent to the specific antigen epitope interaction you are trying to detect. TBST is commonly used for this purpose. It is isotonic to normal saline, in buffered around physiological pH.
Nazik Mansour thank you very much...if I do no use serum with TBST will that influence my results?
In general, serum (same species as the secondary antibody) or bovine serum albumin (BSA) is used for blocking. Sera and BSA can help to prevent unspecific binding to the many hydrophobic side chains of proteins present in the tissue. If you are staining with multiple antibodies, you need to use blocking serum against all used secondaries. If BSA is used, the addition of 0.1–0.5% Triton-X or Tween can help to prevent unspecific binding
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