I have cultured cells which were exposed to a certain drug in a 24 well plate at different conc and time points. At the end of the experiment, I used Trizol for RNA isolation. I removed media from each well first, then added Trizol to each well, pipetted up and down and added those lysed cells to fresh tubes. Then I followed RNA isolation protocol. Briefly, I added chloroform and centrifuged the tubes. This separated the mixture into aqueous, interphase and organic phase.
At what point I should have saved supernatant for protein analysis for ELISA?
This has a protocol for Trizol-extracted protein.
https://www.creative-diagnostics.com/total-protein-extraction-by-trizol.htm
Hey Anie,
all cytokines, which were secreted by your cells during the different conditions are in the supernatants. You can just transfer your supernatants in a 96-V-shaped plate and centrifuge for 5 min at 650g to pellet debris and then transfer the fluid to a Flat-Bottom-Plate for storage at -20°C (to later analysis with ELISA for example).
I think there is a big difference between mRNA levels of cytokines, cytokines that are build but not secreted (that would be your lysed cell fraction) and cytokines that are indeed released by the cells.
Cytokine-specific mRNA does not necessarily mean the protein was build (mRNA can be stored without protein building), cytokines can be stored in the cytosol in vesicles without secretion and, finally, cytokines can be secreted after a stimulus. This is in my opinion the most important and final step, because extracellular signaling is the main purpose of cyto- and chemokines.
I hope that helps you.
All the best,
Marc
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