Hi everyone -
I'm setting up an ELISA for the first time and mostly figuring it out on my own. I'll be analyzing secreted proteins in cell culture supernatants. Looking through protocols and guides, I'm not seeing anything about normalizing the measured analyte levels to the number of cells (i.e. as is done with housekeeping genes in PCR). My cells will have been seeded several days before, and for all I know, my treatments may have affected their proliferation.
Am I missing something? Is there a control typically included that accounts for this? Or, is it usually not taken into account? All I can think to do is run an MTT (I'm not confident I could trypsinize my cells after the treatment) and normalize to that, but that goes for metabolism and not truly cell count. I don't have the funds or time for something like BrdU. Quantifying the total protein in the supernatant seems sketchy, since maybe most of it would be coming from the FBS anyways. And if no one's doing it, there's probably a reason why...can somebody explain? Thanks.
Hi,
From my little experiences I am trying to answer your question (if I have understood your question properly)-
To normalize the results of ELISA, it would be better to do the followings:
1. Please maintain cells without treatment (hope it is already in your design) which could be considered as control.
2. Maintain a separate set(s) of cells and use those cells for trypan blue dye exclusion method (live cell count) and/or prestoblue/ alamar blue/ MTT assay (cell viability/metabolic assay).
Finally, you may normalize your ELISA result(s) as targeted protein(s) conc./million cells. Following normalization, like PCR you may present your result(s) as up-/down- regulation of targeted protein(s) under certain conditions. Or simply you may present your cell proliferation/viability data and ELISA separately while explaining possible correlation between them under certain conditions (treatments).
Hope it would be helpful. Thank you.
Best wishes,
NH
I think you should divide the results of treated by non_treated supernatant. Maybe its be useful if you divide the absorption of supernatant in those conditions
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