I will do a GSIS in min6 cells and normalize the results to total protein, after taking the supernatant for high glucose stimulation do I need to wash the cell with PBS before adding lysis buffer to the cells?
Absolutely Yes
You can also use RPMI nutrient media to wash cells
Best wishes
I would absolutley recommend washing with PBS rather than media.
If you normalize your data to total protein content, and the media you had the cells in contains protein, it will screw up your data. The amount of medium that stays in between the cells will not always be the same, so you will definitly change your results if you do not wash with PBS!
If you want to, you can try it out:
Take the some of your cells, split in equal portions, wash half with PBS and half with RPMI (or don't wash at all) and check the total amount of protein. That way you can judge the impact of washing.
Hi there,
Cell wash is compulsary prior whatever you do with it. Just to be able to normalize the results to the cell number per sample and to get sure there is no residual medium effect on them.
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