Hello, recently I have been detecting caspases cleavage with western blot. But I found caspases cleavage in control/untreated group, what can be the possible reasons?
I used QGP-1 cell line. The cells look healthy under microscope(not detached from flask, cell morphology is normal).
Hello,
The reason for cleavage in control sample might be the protein sample preparation and protease inhibitor. Try to run the western on same day of protein isolation to avoid the cleavage and protein degradation.
Good Luck!
Hi Sangappa Chadchan,
Thank you for your quick answer.
Normally I prepared samples immediately after protein extraction with addition of loading dye and heating up which will eliminate any possible protease activity. Then samples were kept in -20 until the next day of usage.Do you still think sample preparation is a problem? I will check if it is protease inhibitor problem during extraction step.
Also you can take more precaution during protein isolation from cells, by performing the protein isolation step on ice.
Always terminate the experiment before reaching the cells to 95% confluence and equal distribution of cells during plating also helps in minimizing the later cell clump formations, which might lead to some cell death.
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