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How does high salt extraction of nuclear proteins work


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#1 scientistI

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Posted 15 February 2010 - 03:18 PM

Hi all. I have a conceptual question. How does high salt buffer extract proteins from the nucleus? Do you know the mechanism of action?

Also, how does RIPA buffer extract proteins from the nucleus...because of the NP40 and sodium deoxycholate? Thanks!

#2 bob1

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Posted 15 February 2010 - 04:11 PM

The same way that any salt extraction of proteins works. Of course it relies on you having a nuclear fractionation first if you only want the nuclear proteins.

#3 scientistI

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Posted 16 February 2010 - 09:37 AM

The same way that any salt extraction of proteins works. Of course it relies on you having a nuclear fractionation first if you only want the nuclear proteins.


How does regular salt extraction of proteins work? My question is how it gets the proteins from inside the nucleus to outside the nucleus such that the proteins wind up in the supernatant after the separation spin. Thanks.

#4 HomeBrew

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Posted 16 February 2010 - 12:52 PM

I guess the confusion is that most of us are familiar with concentrating proteins using high salt buffers, which cause proteins in aqueous solution to be less soluble because there are less water molecules available to interact with the proteins -- since they're busy keeping the salt dissolved -- so the proteins interact with each other and fall out of solution. But this is generally true of any protein solution -- usually there's a step preceding this that in some way enriches the solution with proteins from a particular compartment -- in your case, from the nucleus.

Are you following a particular protocol or using a particular kit?

#5 scientistI

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Posted 16 February 2010 - 01:24 PM

I guess the confusion is that most of us are familiar with concentrating proteins using high salt buffers, which cause proteins in aqueous solution to be less soluble because there are less water molecules available to interact with the proteins -- since they're busy keeping the salt dissolved -- so the proteins interact with each other and fall out of solution. But this is generally true of any protein solution -- usually there's a step preceding this that in some way enriches the solution with proteins from a particular compartment -- in your case, from the nucleus.

Are you following a particular protocol or using a particular kit?


I'm using the Pierce Nuclear Cytoplasmic Extraction Kit, which is based on NP40 cell lysis and high salt nuclear extraction. What I'm unclear on is how the salt removes the proteins from the nucleus. In their protocol, the transcription factors remain soluble in the supernatant while nuclear debris pellet.

#6 HomeBrew

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Posted 16 February 2010 - 08:33 PM

According to the kit instructions:

Addition of the first two reagents to a cell pellet causes cell membrane disruption and release of cytoplasmic contents. After recovering the intact nuclei from the cytoplasmic extract by centrifugation, the nuclei are lysed with a third reagent to yield the nuclear extract.


So, it's a two-step lysis procedure. The first steps lyse the cell membrane and allow separation of the cytoplamic proteins from intact nuclei. With the bulk of the cytoplasmic proteins removed, the nuclei are then lysed to liberate the proteins therein. I'm not sure what you mean by "how does high salt buffer extract proteins from the nucleus". The proteins are liberated from the nucleus by lysis.

#7 laurequillo

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Posted 17 February 2010 - 08:54 AM

According to the kit instructions:

Addition of the first two reagents to a cell pellet causes cell membrane disruption and release of cytoplasmic contents. After recovering the intact nuclei from the cytoplasmic extract by centrifugation, the nuclei are lysed with a third reagent to yield the nuclear extract.


So, it's a two-step lysis procedure. The first steps lyse the cell membrane and allow separation of the cytoplamic proteins from intact nuclei. With the bulk of the cytoplasmic proteins removed, the nuclei are then lysed to liberate the proteins therein. I'm not sure what you mean by "how does high salt buffer extract proteins from the nucleus". The proteins are liberated from the nucleus by lysis.


Actually what I understand (and with the protocol I use: Buffer C only has 400mM NaCl and no detergent) you get your proteins without lysis. Thats the point of the high salt buffer, to be able to extract the proteins without nuclei lysis

Edited by laurequillo, 17 February 2010 - 08:56 AM.

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#8 HomeBrew

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Posted 17 February 2010 - 05:17 PM

Maybe Google steered me wrong -- I may have linked to the wrong instructions. Having never used the kit, I googled "Pierce Nuclear Cytoplasmic Extraction Kit" and grabbed the manual from the piercenet.com page that came up...

#9 laurequillo

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Posted 17 February 2010 - 08:38 PM

I dont know, I guess there are different protocols, but with the "classic one" (I dont use a kit), once you remove the citoplasmic proteins, you wash and then you add a high salt buffer without detergent (my buffer C is 400mM NaCl, EGTA,EDTA, betamercaptoethanol...). With that method you are supouse to be able to get the nuclear proteins without nuclei lysis.

Actually I dont really know the difference without that method and one in which you use detergent to get the nuclear extract. It is just that without detergent is more clean??

Edited by laurequillo, 17 February 2010 - 08:53 PM.

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#10 laurequillo

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Posted 18 February 2010 - 12:50 AM

Here the explanation from "Current protocols in molecular biology":
"PREPARING NUCLEAR EXTRACTS
To prepare nuclear extracts, tissue culture cells are collected, washed, and suspended in
hypotonic buffer. The swollen cells are homogenized and nuclei are pelleted. The
cytoplasmic fraction is removed and nuclei are resuspended in a low-salt buffer. Gentle
dropwise addition of a high-salt buffer then releases soluble proteins from the nuclei
(without lysing the nuclei). Following extraction, the nuclei are removed by centrifugation,
the nuclear extract (supernatant) is dialyzed into a moderate salt solution, and any
precipitated protein is removed by centrifugation"
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