I have a lyophilised biotinylated peptide which is of neutral charge and more hydrophobic than hydrophilic. What is the most suitable substance to use to ensure that it is still viable for an ELISA?
Hi Harry, I have used a biotinylated lyophilised peptide for an ELISA and I dissolved it in sterile water (as stated in the manufacturers instructions). I don't want to give you too much specifics because getting in touch with the manufacturers is best as they usually have done stability studies. Good luck.
The simple answer: coating of your ELISA plate with (Strept)Avidin, 1 ... 5 µg/mL in a 0.1 mol/L carbonate buffer pH 9.6, overnight at 4 °C, washing with PBS 3 - 4 times, removal of most of the residual fluids. Incubation with your peptide, concentration at about 1 µg/mL for 15 min ... 2 h (use the longer time, this avoids "hecticness"). washing with PBS 3 - 4 times (no detergents up to this point), removal of most of the residual fluids. This will simply bind the peptide to the solid phase avidin.
The question about the function of your peptide is still open. You need to have some idea about the binding of the biotin onto your peptide, N-terminal, C-terminal, or on any free residuals (Cys, Arg, Lys, ...) and the distance of the Biotin to your "active center" of your peptide.
You can control this by the selection of the biotinylation site.
Hai Harry,
I think it dissolved in deionised water.Keep it at room temperature(25-30*C) for half an hour before conjugation with Streptavidin.
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