I am doing an experminent on human monocyte derived macrophages to see profile of cytokines after stimulation with a MoAb by doing ELISA on both supernatant and lysate. Is it necessary to measure total protein before running ELISA to confirm that wells with the same treatment conditions have the same amount of total protein (especially the lysate)? If yes, how should corrections/adjustments be made after measurement of total protein?
Dear Mr TAREG,
Why doing an ELISA for cell lysate to a cytokine profile? The supernatant is enough
Hi, to get accurate results, you must normalize your protein-of-interest quantity vs biological material amount. You can do it with total cellular proteins, total cells number or DNA for example. Results are then exprimed in ng or µg of prot-of-interest/ million cells or µg of total prot or µg/ng of DNA; If you don't, you cannot have any idea on the origin of the observed differences between samples , which is likely to lead to misinterpretation of ELISA results.
Thank you Guillaume Vidal for the useful answer. One further question, though. I need a little more explanation on the point " You can do it with total cellular proteins, total cells number or DNA for example," particularly the 'total cells number' part. Is normalization achieved if I have the same number of cells in each well? In my experiment ,for example, 20k cells per well.
@Faez Amokrane Nait Mohamed, that is an important question. I don't think I have a confident answer to it and I don't know is the best answer I have right now. However, although cytokines are generally secreted to the supernatant; some are retained largely in the intracellular compartment. My target protein has an intracellular variant and I want to see the effect of the MoAb stimulation on both secreted and intracellular variants by comparing MoAb stimulated cells with un-stimulated and isotype stimulated cells. This is my take on it in relation to my particular experiment. But to the larger question as why ELISA should be done on cell lysate, I hope people with experience on the topic can provide a detailed answer.
You want to study the difference between secreted and intracellular variant of your target protein. It's OK, so it is very important to measure the total protein amount on your cell lysate.
Best regards
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