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Cytoplasmic/nuclear fractionation with NP-40 then RIPA buffer combination?

Nuclear Cytoplasmic Cell fractionation Protein NP40 and RIPA

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#1 5280

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Posted 28 October 2015 - 07:36 PM

Hi, 

 

I was wondering if this simple cytoplasmic / nuclear fractionation would work for mammalian epithelial cells. Use NP40 lysis buffer on the cell pellet for cytoplasmic fraction then pellet nuclei. Lyse nuclei with RIPA and spin down to obtain nuclear soluble fraction. Lastly, nuclear the remaining pellet can be sonicated or Mnase digested for obtaining the chromatin fraction. 

 

Any suggestions or obvious problems that I am missing with this approach?

 

Thanks!



#2 Curtis

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Posted 28 October 2015 - 11:43 PM

the RIPA that you want to use for nuclei lysis must have sodium deoxycholate.

But I wouldn't do it this way. NP40 is still a strong detergent. I would use other methods of fractionation. 



#3 5280

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Posted 29 October 2015 - 08:27 AM

Yes, the RIPA has sodium deoxycholate. Maybe the NP40 buffer could be used at 0.5% if 1% if it is too high %? I currently use the Pierce NE-PER fractionation kit but I thought the NP40/RIPA approach might be cheaper and quicker. 

 

NP40 lysis buffer

25 mM Tris-HCl pH 7.4

150 mM NaCl 

1 mM EDTA 

1% NP-40 

5% glycerol 

 

RIPA

1x PBS
1% NP40
0.5% Sodium deoxychlolate
0.1% SDS
 

 

Plus the addition of protease and phosphatase inhibitors to both. 






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