We have been trying to incorporate AHA (L-Azidohomoalanine) into the cells, in comparison to normal Methionine, and following click-it reaction based on published protocols to test the incorporation using western blot with Streptavidin. Unfortunately it looks as if both samples were labelled (signal in both WB samples). Does anyone have some experience with this or can anyone suggest anything to differently?
Hi,
I've been using a commercial kit to detect labeling, clicking the labeled-proteins to TAMRA and fluorescence imaging. I see no background in the unlabeled samples.
Did you have time so far to try something different in your samples?
Best,
CM
Hey Carlo,
We've been trying to incorporate the AHA and do the click-it reaction using "home-made" kit. After we saw no difference between AHA and Met samples for western blot analysis, we've been trying to explore the AHA incorporation into proteins in comparison to the Met, using MS. It seemed like the incorporation was very low (around 1%) after 4 hours of labeling. The observation repeated itself several times (tried different times of labeling, different AHA concentration, other cells, etc.). Did you try to starve the cells from Met by a special technique? Did you try to explore the incorporation of the AHA into your newly synthesized protein in other means?
Thank you in advance!
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