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Is mCherry denatured by Trypsin or RIPA buffer?


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7 replies to this topic

#1 labmate1000

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Posted 01 September 2011 - 04:38 PM

Hi everyone,

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!!!

#2 bob1

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Posted 01 September 2011 - 08:09 PM

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?

#3 Rsm

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Posted 05 September 2011 - 05:31 AM

I would think that your Trypsin may affect mCherry signal, particularly when you lyse the cells. Do you use protease inhibitors?
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#4 labmate1000

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Posted 06 September 2011 - 12:53 PM

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?

#5 bob1

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Posted 06 September 2011 - 03:20 PM

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.

#6 labmate1000

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Posted 08 September 2011 - 08:43 AM

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?

#7 bob1

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Posted 08 September 2011 - 09:54 PM

In that case the trypsin won't be such a problem. However, there are still proteases inside the cells which will be!

#8 labmate1000

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Posted 14 September 2011 - 10:47 AM

Thanks! I will definitely use protease inhibitors next time to see if I see different results.




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