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Whole cell lysate including nucelar proteins?


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#1 mjlm3

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Posted 17 February 2010 - 07:04 AM

Hi!

I want to prepare a whole cell lysate (HEK cells) for an immunprecipiation experiment. The lysate should contain cytoplasmit AND nuclear proteins (specifically, I'm interested in a transcription factor which might be nurlear-localized).

Standard protocols for whole cell extracts use hypotonic Tris-buffers, will such a lysate contain nuclear proteins as well? I'm just wondering because protocolls for nuclear ectracts usually use a high salt extraction or something harsh like that.

Thanks,
Mat

#2 laurequillo

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Posted 17 February 2010 - 07:38 AM

Hi!

I want to prepare a whole cell lysate (HEK cells) for an immunprecipiation experiment. The lysate should contain cytoplasmit AND nuclear proteins (specifically, I'm interested in a transcription factor which might be nurlear-localized).

Standard protocols for whole cell extracts use hypotonic Tris-buffers, will such a lysate contain nuclear proteins as well? I'm just wondering because protocolls for nuclear ectracts usually use a high salt extraction or something harsh like that.

Thanks,
Mat


Sure you will have both
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#3 mjlm3

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Posted 17 February 2010 - 12:59 PM

Sure you will have both


Thanks for the answer. Why is it then that protocols for the extraction of proteins from the nuclear fraction usually use high salt concentrations etc.? Is that just to get the tightly chromatin-bound proteins (histones etc.) into solution?

#4 laurequillo

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Posted 17 February 2010 - 08:46 PM

Sure you will have both


Thanks for the answer. Why is it then that protocols for the extraction of proteins from the nuclear fraction usually use high salt concentrations etc.? Is that just to get the tightly chromatin-bound proteins (histones etc.) into solution?


Actually with the protocol I use, when you extract the nuclear proteins you just add a high salt buffer without detergent, and the point is to get the proteins from the nucleus without lysis. I just was discussing that point in another post. I guess you just can add some buffer with detergent, after de citoplasmic extraction, and lysate the nucleus and you will get your proteins as well. I did not think about that before, and I a not really sure about the differences.

But for sure if you use a "normal" lysis buffer you will get nuclear and citoplasmatic proteins. The only thing is that you will not get the insoluble fraction (there are some proteins that are quite insoluble, they remain for example in some nuclear bodies, like pcG bodies). To get those too you should do a SDS lysis. Just boil the samples for 2min in SDS buffer (laemmli buffer) 1x, the sonicate 2x 20sec and then boil again 5min.

Edited by laurequillo, 17 February 2010 - 08:59 PM.

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