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help please for fractionation!!
Posted by: aniu1229 (IP Hidden, New member, 8)
Date: July 11, 2005 09:06AM
I have a problem with my subcellular fractionation. I am checking the localization of a protein. It has been reproducibly confirmed to be in nucleus by immunofluorescence, but when I did fractionation, most of the protein always stay in cytosol. I have used tubulin as cytosol marker, and PARP or B23 as nuclear marker to prove that fractionation itself worked well.
so I want to ask friends here, whether you know and possible factor during fractionation will affect protein localization during the experiments of fractionation. when I harvested cells, I either trypsinized cells or detached them just simply by pipetting. but the results did not change. also I noticed that, with one membrane, different nuclear marker may give different result. for example, cytosol fraction was shown to be very clean when blotted with PARP or histone antibody, but when I blotted with another kind of nucleolin Ab (home-made antibody form some other labs), I could see cytosol fraction was contaminated by nuclear fraction a little bit. so do you think it reasonable? so Would you please tell me, why IF and fractionation will give different results, and whether it is possible to solve this problem and how? I suppose that maybe my protein is loosely attached to some nuclear protein. so it may go to cytoplasm during fractionation, but we do not know what is the factor to affect that. Thanks in advance!
Re: help please for fractionation!!
Posted by: mitolab (IP Hidden, Senior member, 89)
Date: July 11, 2005 09:48AM
hi,
Is the staining in the nucleus or it is paranucleus? Are you using the same antibody in both staining and immunoblot? Depending on which method are you using to extract nuc/cyto fraction, you may get different level of cytosolic protein contamination in your nucleus fractionation. But if your staining shows exclusively nuclei staining then your immunoblot should reflect this subcellular localization. However if it is paranucleic staining it is possible the protein is in ER or golgi apparatus. It is possible to distinguish by co-staining with ER/golgi antibody. My two cents of opionion.
Re: help please for fractionation!!
Posted by: aniu1229 (IP Hidden, New member, 8)
Date: July 11, 2005 10:17AM
Hi, mitolab, thanks a lot for the information!
From IF result, the protein spreads allover the nucleus, but not paranucleus. and I also use the same anitbody for both IF and IB. so I do not know what is the problem. but I will try to do costaining with ER/golgi ab in case of any new findings. By the way, do you by chance have some good protocols for fractionation? Thanks!
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