I am performing an ELISA to quantify TNFa and perforin in PBMC culture supernatant after 48 hour stimulation with peptide. It's my first time performing the assay, so I lack speed and experience. After the supernatant was obtained from my PBMC culture, I let it sit at room temperature for about 1 hour and 40 minutes before finally transferring it to a -80 freezer. I was pre-occupied with another experiment that I was trying to complete at the same time.
How badly will my ELISA assays be affected? Are my cytokines severely degraded?
Thanks for your help!
Hello Philip Samaan
Storage of tissue culture supernatant containing protein of interest at room temperature often leads to protein degradation and/or inactivity. After obtaining the supernatant from PBMC culture, instead of letting it sit at room temperature, you should have kept the supernatant on ice or in the refrigerator at 4 degree C which I assume must have been within reach from your workstation. After completion of your other experiment, you could have transferred the supernatant to -80-degree C. At room temperature proteins start degrading with time.
As far as TNFa is concerned, it will be affected if you let it sit at room temperature for more than 1 hour. Please refer to the articles attached below.
https://helda.helsinki.fi/server/api/core/bitstreams/bd5ff099-6683-4aca-9a1d-386bfd7db65a/content
https://www.sciencedirect.com/science/article/abs/pii/S0306453020304923
Perforin is not a cytokine. It is a 60-70kDa glycoprotein which is a cytotoxic molecule. I am not certain about the stability of perforin at room temperature, but I am sure that it would also start degrading at room temperature with time.
Best.
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