ELISA information

Wed Apr 5 17:01:57 EST 1995

> Our
>antigen is extracted using a 4M urea, 5% mercaptoethanol, 0.2% Triton X-100
>buffer and the protein concentration is approx. 1.25 mg/ml.  Using this full
>strength or diluting in different buffers with various pH and buffer
>concentration has not been effective in generating a signal. 

1.25 mg/ml - I assume that you have a hole bunch of proteins
over there. First, I would try to dilute the extraction
mixture to a normal coating concentration 10-50-100 mkg/ml.
Then I would dialyse against low ionic strength (10-20 mM)
 buffers of DIFFERENT pH. For any given antigen there an optimum pH for the
coating buffer, and you cannot predict it.

One thing is for sure:  Urea and Triton X-100 are inhibitory
for coating.

andrei.popov at bbsrc.ac.uk

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