[Protein-analysis] Re: question (Kate Chabarek)
clement at bio.mls.eng.osaka-u.ac.jp
Fri Jul 14 20:19:59 EST 2006
Do you know the amino acid sequence of your protein? If it has much more Phe
than Trp or Tyr, then it should have a max absorbance value at 260 (your
protein might be pure). Otherwise, it may be contaminated with DNA or RNA.
If you cannot see the DNA in agarose, then the nucleic acid contaminants may
be of low MWs (try PAGE stained with Et-Br to confirm). Try dialysis using
tubes with fixed MWCO to remove your contaminants, or try filtration (we use
centriplus from millipore in our lab).
Hope this helps.
> I am trying to isolate 100 ug of a transcription factor in order to
> determine its phosphorylation sites.
> I unsuccessfully tried a lot of different protocols and ended up, crude as
> it sounds, doing a huge immunoprecipitation under denaturing conditions.
> I get one band of the correct size by silver staining, zinc-imidizole
> staining, and Western blotting. Hooray-except that the A260 of the
> HPLC-separated sample is about 60% larger than the A280. I ran an agarose
> gel and don't see any DNA there. Also, I can't confirm its identity by
> mass spec. I don't know what this band could be if it isn't my protein of
> interest~it doesn't match the size of anything else in my
> immunoprecipitation. Because it is very low-abundance and has no enzyme
> activity it is hard to quantify. I am wary of sending it out since I can't
> confirm what it is and because of the weird A260 result.
> Has anyone had this experience, and what did you do?
> Thank you.
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