Hello.
I wondering i must do add protease inhibitor(like a PhoSTOP,,) before ELISA experiment?...
My sample is human plasma. Before start ELISA experiment, plasma samples are distributed 30ul, 60ul volume.
because I forgot to add protease inhibitor. TT..
My samples are thawed just 1 time now.
maybe 1-2 times more elisa experiment planning now.
Help me plz. Thank you
It very much depends on your analyte you want to determine. Many ELISA experiments are performed without addition of protease inhibitors.
As it's plasma, not serum, there might a chance that some proteases are liberated from broken cells (freeze-thaw should do that, unfortunately).
If they may be doing any damage or not depends on the setup of your ELISA. Can you tell us more about it (which analyte, sandwich or not, dedicated assay buffer, homemade or commercial, how were the serum samples obtained)? What is PhoSTOP composed of (actually, it rather sounds like a phosphatase inhibitor to me. In that case, you also could try NaF plasma instead or add NaF up to 100mM).
To exclude (or to confirm) any worries, you should try to validate your system and the data: The next time, you run an assay, compare samples that have been freeze-thawed with an aliquots that haven't and where protease inhibitors (the right ones!) have been added (and combinations thereof to find the sensitive spot).
If the sample is human saliva does protease inhibitor need to be added before ELISA
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