Protocol Online logo
Top : Forum Archives: : Protein and Proteomics

cell fractionation plus IP - (Sep/27/2005 )

Pages: 1 2 Next

Hi there,
I am trying to study the interaction of my protein w/another by cell fractionation, followed by IP.
I am having a couple of problems I hope you guys can help me solve.
1) If i don't spin my cytosolic fraction before adding the antibody, I see a nice co-IP. Also, I see an interesting tubulin co-IP, consistent with what I see in IF, shape-wise. If I spin the sample before adding the ab, then most of that association is gone. What should I do in order to be sure that that association is specific (nonspecific Abs will also bring down this macromolecule)? How are associations with the cytoskeleton studied? only by IF?
2) I add NaCl to my cytosolic fraction to reach 150 mM before the IP (it is at 10 mM for fractionation). Is that necessary for the IP? How can IPs be affected by this difference in salt? I do the opposite with the nuclear fraction, I dilute it to 150 mM from 420mM NaCl.

Thanks!
d

-dapo-

hi,
i understand you're doing cytosolic/nuclear fractionation? thing is that in your buffer cytoskeleton fraction may not be soluble. do you need to fractionate? what's your buffer composition in the fraction you IP from? you may try eg to increase your detergent concentration.
if it goes for NaCl, i think salt concentration is important with ip. 150mM is the min. i ever use. you can actually increase the salt, first to 200mM.. then maybe higher (depends what ealse is in your buffer). salt gives ions so it helps to decrease the backround.

-Jusu-

Thanks for answering, Jusu.
Yes, I am trying to fractionate Cyt-Nucl. I do need to fractionate. The buffer composition in the Cytosolic fraction is: 10 mM hepes pH 8, 10 mM KCl, 1.5 mM MgCl2 and I add 5M NaCl to final concentration of 150 mM before the IP. I could eventually add NP40 to 0.5% -1% to try and solubilize the cytoskeleton?
Thanks a lot

-dapo-

Can you get away with using a cytoplasmic/nuclear fraction kit say from Active motif and then do an IP?

-Pria-

Pria, not really. B-cells are 99% nucleus, and that proved to be a problem using kits, because the nuclei would break easily and leak into the cytosolic fraction

-dapo-

QUOTE (dapo @ Oct 3 2005, 04:53 AM)
Thanks for answering, Jusu.
Yes, I am trying to fractionate Cyt-Nucl. I do need to fractionate. The buffer composition in the Cytosolic fraction is: 10 mM hepes pH 8, 10 mM KCl, 1.5 mM MgCl2 and I add 5M NaCl to final concentration of 150 mM before the IP. I could eventually add NP40 to 0.5% -1% to try and solubilize the cytoskeleton?
Thanks a lot


yes i would try that. either with NP40, or triton.
so, you spin your cytosolic fraction to remove the possible remaining nuclear conatamination? if yes, i would spin but at low speed - just like for nuclei spin. when you'll spin faster it's possible that you may loose the cytoskeleton as well.
besides, cytoskeleton is hard to sulobilize. if triton or something will not help, you may consider adding a little bit of sds.. like 0.1%? or even lower for a strart and just play around with the conditions. dont really know how strong is your interaction.
good luck

-Jusu-

If you use concentrations as high as 0.5-1% of NP40 or Tx-100 you're gonna coextract nuclear soluble pools as well. Is it possible to use some crosslinking approach previous to solubilizing the proteins by strong conditions?

-miguelon-

QUOTE (miguelon @ Oct 7 2005, 11:05 AM)
If you use concentrations as high as 0.5-1% of NP40 or Tx-100 you're gonna coextract nuclear soluble pools as well. Is it possible to use some crosslinking approach previous to solubilizing the proteins by strong conditions?

of course, that is why the detergent should get there after removal of nuclei, and before spinning cytoskeleton. what kind of crosslinking do you have in mind? dont you think that even if possible it would make the interaction studies a bit weird?

-Jusu-

Sorry, maybe had not read properly your reply, you're suggesting to add the detergent after the fractionation step. Is a good idea.
About the crosslinking, I have not tried by myself, I was asking you whether you knew something about it. I think that some chemical crosslinking approaches are reversible, like PFA. Maybe you could crosslink direct interactions in that fraction, in order to apply harsher solubilizing conditions and keep stable your target interaction during that step? Has anybody tried that approach? Is it only suitable for in vitro studies?

-miguelon-

Thanks guys.
I'll try the NP40 addition AFTER spinning down the nuclei.
I've never tried crosslinking, but I think it works, although it's not ideal....

D

-dapo-

Pages: 1 2 Next