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Growth-arrest by serum starvation


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#1 R.Lolo

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Posted 23 March 2009 - 06:22 AM

Dear All,
I'm trying to investigate how does glycosylation change interaction of YY1 with its molecular partners (part of my master project).
My plan is to tranfect HEK293 cells with my constructs (calcium phosphate method) and then grow them in high-glucose. According to publication I found I should also induce growth-arrest by serum starvation when using high glucose concentration. i don't entirely understand why. If I transfect my cells and keep them in high glucose, serum free medium how would that effect proteins synthesis process? I need to investigate the role of glycosylation which is postranlational process. My plan is:
1. 0h transfect cells
2. +12h - replace medium (full medium)
3. +36h - replace medium (high glucose, serum-free)
4.48-60h conduct and experiment.

What do you think about the schedule? I wouldn't like to affect protein expression by starvation (do you know how much does starvation inhibit synthesis?). On the other hand I think 12-24 hours is enough to stimulate hehamine pathway and to glycosylate proteins.

Thanks in advance,

R. Lolo

#2 bob1

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Posted 23 March 2009 - 02:16 PM

Serum starvation forces the cells (mostly) to into G0 arrest. The cells are still alive and functioning properly, they just can't cycle. The should still produce proteins etc, though if you leave them in the serum starvation state for too long they will undergo apoptosis.

#3 R.Lolo

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Posted 23 March 2009 - 02:31 PM

Thank you. I found and information that my protein of interest interacts with some of the major cofactors only during G0. Supposedly the authors intented to put cells into G0 then.
Thanks a lot.

Rafal




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