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over trypsinization


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#1 scientistI

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Posted 31 March 2010 - 02:48 PM

Hi, I read on the forums that over trypsinization will lead to cell lysis. People say that the cells break open, release DNA, and clump. But, I treated BJ fibroblasts with 0.25%Trypsin-EDTA for 30min at 37 degC and didn't really notice a difference in pellet size compared to BJ fibroblasts treated with Accutase for 10min at 37degC. I washed the cells a few times in PBS and counted them...the numbers were similar. The trypsinized sample did have a greater number of blue nuclei, but the cells themselves looked intact? So what exactly happens when you trypsinize cells for 30min? Does it pop open, permeabilize, or what? I'm trying to strip off proteins bound to the outside of the cell, so would acetic acid be a gentler method? What would be the most gentle yet most effective method? Thanks!

#2 bob1

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Posted 31 March 2010 - 03:18 PM

Your blue cells are dead cells - they will not grow if you tried to plate them out again, so the numbers are probably not similar between your long trypsinisation and the shorter one.

Trypsin digests proteins at lys or arg residues, it is pretty non-specific so it basically digests the cell structure and proteins sticking out of the membrane. I would have a look for secreted protein isolation protocols.

#3 jah

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Posted 01 April 2010 - 04:51 AM

You might try harvesting your cells with 0.53mM EDTA only...much gentler and does not digest proteins.

#4 scientistI

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Posted 01 April 2010 - 01:12 PM

Hmm I'm actually trying to get rid of the proteins that are stuck on the cell surface. It's like when you're doing a radiolabeled ligand for a receptor and you want to strip off the uninternalized radioligand. Is there a standard process people do for that?




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