I am trying to measure protein synthesis rate of a particular secreted protein that is very hard to see in western blot. However we have a good stablished method for ELISA detection.
My question is: Can I perform a Click-IT AHA experiment (for incorporation of L-Azidohomoalanine in all nascent protein), then capture my protein of interest by ELISA and read (A) Newly synthesised protein of interest & (B) total protein of interest in the same sample?
Thanks in advanced
Shamsher S. Kanwar the steps is would follow (very summarised) are:
1. Treat my cells following Click-iT AHA protocol to incorporate AHA in nascent peptides. Incubate for 6h to allow for protein synthesis and release. Collect supernatant for ELISA and protein for other aplications.
2. Coating ELISA plate with primary ab against my protein of interest (POI)
Blocking ELISA plate
Incubating supernatant sample in the ELISA plate
- At this point I expect my POI to be bound to the plate
Incubating with Detection reagent against labelled AHA residues.
Conjugate with Streptavidin, develop and measure absorbance.
Theoretically it should work, but I cannot find anyone who has done click-iT AHA in secreted proteins, so I don't know if using the detection secondary antibody for AHA is compatible with ELISA as it involves different processing than the widely validated immunofluorescent application.
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