Is mCherry denatured by Trypsin or RIPA buffer? - (Sep/01/2011 )
I am currently transfecting cells with mCherry and lysing the cells to determine the fluorescence of mCherry. However, it has just been brought to my attention that using trypsin to lift my cells and/or my lysis buffer may affect my mCherry protein. I can't find anything in the literature about it, so I was hoping someone may have some knowledge in this? Thanks!!!
The trypsin should only affect the exterior of the cell, but RIPA might denature it a bit, though it shouldn't as RIPA is often used for IP and as such should be isotonic and non-denaturing.
Couldn't you determine the fluorescence on cells on a slide?
I would think that your Trypsin may affect mCherry signal, particularly when you lyse the cells. Do you use protease inhibitors?
Thanks for the responses.
Is it possible to determine the fluorescence on a slide after I have lysed the cells?
No I am not using protease inhibitors. Do you mean I should use protease inhibitors when I'm lysing my cells with the Ripa buffer?
No it isn't possible to check it on a slide after lysis. You could put some through a fluorescence plate reader with appropriate controls though.
You should always use protease inhibitors when lysing, this is to stop your proteins from degrading quite so quickly! Unless you have washed the cells very well there will still be some trypsin with the cells after lifting, and they also contain native proteases.
After trypsinizing, I normally wash the cells 3x with PBS after I pellet the cells. Do you think the trypsin will still be a problem?
In that case the trypsin won't be such a problem. However, there are still proteases inside the cells which will be!
Thanks! I will definitely use protease inhibitors next time to see if I see different results.