ANDREI.POPOV at bbsrc.ac.uk
Wed Apr 5 17:01:57 EST 1995
>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
andrei.popov at bbsrc.ac.uk
More information about the Immuno