phosphorylation experiment without using radiation? - (May/01/2005 )
I'm a relatively new graduate student who will soon be looking at phosphorylation of proteins. I'm just wondering if there are any good methods out there that does not require the use of radiation. Ultimately if that's the most useful method, I will use it, however, if there is a way to avoid using any radiation (esp. P22), I would rather go for that.
Any comments would be appreciated. Thanks in advance.
i don't really know what kind of phosphorylation study you want to make, but there are ones without radiactivity.
You can study the protein by mass spectrometry and detect phosphorylation state.
You can use antibodiers specific for the phosphorylated form....
I'm also dealing with phosphorylated forms of proteins (or I try to).
If you have an antibody against the protein you investigate you can perform 1D IEF gels followed by western blots (see my current post). The blotting effiency is not very high because the proteins in the gel are not charged but I try to optimize (washing with SDS). With this method you can detect different phosphorylated forms of that protein. And if you have a suggestion about the respective kinase and are able to construct a mutant (I'm working with bacteria...) the phosphorylated band should disapear if your suggestion is right.
With mass-spectrometry the problem is that weak (reversible, "energy rich") phosphorylations are very difficult to detect.
I learned that these antibodies against phosphorylated residues are not very good and very expensive.
Another method is the staining with DiamondProQ which stains phosphorylated proteins in SDS-gels. You should consider that (some people in our group work with that successfully).
hope this helps
can you tell me some more details about this staining of phosphorylated proteins? I`ve never heard of that staining method before and can`t find anything about it in the internet. I`m working on phosphorylation of a potential sensor kinase/response regulator system and up to now I made my assays with gamma-32-P-ATP, but I also would prefer a not radioactive method. Thanks in advance,
the dye is from Molecular Probes
I found a picture of a stained gel on this site: http://www.clarechemical.com/proqdiamond.htm
follow the link to the Molecular Probes site
hope this helps
Thanks for all the help! I'll keep all those techniques in mind!!
danke und Grüße von Rostock nach Greifswald!
danke und Grüße zurück !
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