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double crosslinking in PBS or medium?

chip double crosslinking PBS

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#1 Txaio83

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Posted 19 March 2014 - 06:12 AM

Hi all! I have been doing ChIP experiments for a while, both in tissue and cell lines, and suddenly I was concerned if I was doing it correctly or not. I am using double crosslinking (DSG+ formaldehyde) to immunoprecipitate some coregulators that don´t bind directly to DNA. And I have been using DSG at a final concentration of 2mM, for 30 minutes, and then formaldehyde 1% for 10 minutes (both in PBS). I was wondering whether this time (40 minutes in total) of the cells in absence of any nutrient (because I am not using their cell medium), can make them behave differently or change their protein binding profile. I think for the tissue is correct to do it in PBS, but maybe for the cell lines, could be somehow stressful? Does anybody have any experience? And on the other hand, the DSG seems to be only soluble in DMSO, and after making the stock solution (suggested in the instructions, 25mM), I need to add 800ul for a final volume of 10ml (which is a final concentration of 8% DMSO in the cells!!!) I wonder how toxic this is, because usually for any treatment you don´t use more than 1%. I am having a lot of doubts now... I have done all my experiments like this but I am not sure whether I am doing the correct thing. Any suggestion or comment is very welcomed! Thanks!! 



#2 bob1

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Posted 19 March 2014 - 11:26 AM

The thing about fixation steps is that they kill the cells... this is the whole point of the fixation, it is to stop the cells in a certain state, so I wouldn't be worried about the toxicity at all.



#3 Txaio83

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Posted 20 March 2014 - 01:43 AM

Thanks... I didn´t think about this possibility! huh.png hahaha! Thanks a lot! smile.png 







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