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Synchronization of HEK293T cells for IP

IP cell cycle HEK293T

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#1 ghernandez

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Posted 02 February 2016 - 02:21 AM

Hi there,

 

I want to check whether if an interaction between two proteins is affected by cell cycle. I'll like to perform an IP with cultures enriched in S-phase, G2/M and G0/G1 phases. I've been reading protocols and I've found that serum starvation is easy to perform (2 days in serum-free DMEM and cells should be in G0). My concern is that I've seen this kind of protocol for HEK293 cells and I'll be using the HEK293T.

 

Someone has used serum deprivation as a way to synchronise HEK293T? If it can be used could you facilitate the protocol? Also, how many hours after adding DMEM with serum I have to wait to get cells in S-phase and G2-phase?

 

Thank you in advance,

 

Gonzalo



#2 bob1

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Posted 03 February 2016 - 11:31 PM

Serum starvation tends not to be very effective for most cell lines, usually results in about 70-80% of cells being arrested.  If you do want to go ahead, wash the cells in serum free medium (SFM) several times (I use one quick wash, 3 x 15 min wash and a 45 min wash), then add SFM and incubate for 48 h. You may have to play around a little to get the conditions to work perfectly.

 

As to how long after re-adding serum - how long is a piece of string? It depends on how long they have been without serum, the length of the cell cycle in your cells (remember...lab evolution of cell lines), the medium type, confluence level, etc.



#3 Andrea Fortina

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Posted 16 May 2016 - 01:39 AM

I think that also depend on your cell conditions. In some cases, a lot of cell died after the treatment.


Andrea Fortina

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