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How to pase my Co-IP over the weekend?


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#1 tfcheng

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Posted 27 January 2010 - 07:05 AM

Hi folks,

I am probably have to collect my transfected cells on Friday for a Co-IP exp, now I need to decide what to do is the best. I can:

1. Lyse the cells on Friday, pre-clear lysate and incubate with primary antibodies over the weekend at 4C;

2. Freeze cell pellets on Friday, continue Co-IP next Monday;

3. Keep pellets on 4C, and do Co-IP on Monday.

I usually perform Co-IP without freeze and thaw, fearing that it will cause my proteins of interest to dissociate. But I also wonder if it will cause too much background or deterioration of proteins if I choose (1), please advice, thank you!!


TFC

#2 madrius1

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Posted 27 January 2010 - 10:00 AM

I would really not recommend doing this over the weekend. Unspecific binding, protein degradation or alteration are highly likely to occur. As I suppose from what you say that you can't do the experiment on the weekend, here's what I would do. Let's call it a "quicker than normal IP".

1- Lyse the cells in the morning.
2- Preclear 1-2 hours at 4 C.
3- Ab + Protein extract for 1 hour.
4- Add beads for 1-2 hours.
5- Wash and elute.




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