Use of carbonate buffer to dilute antibody? - (Oct/12/2010 )
Is it necessary to use a carbonate type buffer for capture antibody dilution.
Is it ok to use BSA?
bsa is not a buffer.
yo4trad on Tue Oct 12 19:55:02 2010 said:
Is it necessary to use a carbonate type buffer for capture antibody dilution. Is it ok to use BSA?
The coating buffers most used are 50 mM carbonate, pH 9.6; 20 mM Tris-HCl, pH 8.5; and 10 mM phosphate-buffered saline (PBS), pH 7.2 . I don't think you can use BSA to dilute capture antibody.
You may use it to reduce high background when using polyclonal antibodies (PCA)as capture antibody. Polyclonal serum will result in strong signal, and in this case you may use BSA to preabsorb the serum using competitor protein such as BSA. In ElISA, you can use BSA as blocking agent because of its ability to compete with nonspecific factors in the test sample for available plastic sites.
Hope this helps.
I think if you use BSA to coat your plate you'll end up with a plate loaded of BSA and very little antibody.
I've always used either carbonate buffer pH9.6, or PBS to coat ELISA plates. Just dilute you coating antibody directly in this buffer and add to the wells.
All buffer suggestions are good. Carbonate, etc.. do not put BSA in your coating buffer that contains your antibody. You may also 'shock' your antibody by briefly subjecting it to low pH then diluting in your coating buffer and adding to your wells.
Use BSA or other proteins for blocking uncoated plastic surfaces. there are also commercial blockers which you can try. if you wish to keep your plates dry and for extended periods...use some sucrose in buffer as final block step decant and allow plates to dry inverted. then keep dry in plastic bag with dessicant and refrigerate.