Detection of phosphorylation site - (Jul/23/2010 )

Hello all,
I'm trying to determine if a chloride channel (CLC-3) can be phosphorylated and, if so, at what site. Can anyone give me any suggestions on how I could do this? The only thing I can think of is to pull out the protein using antibodies, doing an acid hydrolysis to separate the amino acids and running it through a gel to detect any phosphorylation. I think this is a pretty old and inefficient method, are there any other methods out there that are better than this? Thanks in advanced.

Tim

You can run lysates on gels and detect with phospho specific antibodies, if available. YOu can IP with an antibody against the protein (preferably with an antibody that is not near potential phosphorylation sites as phosphorylation can interfere with antibody binding), then run on a gel stain with coomassie and cut out the band, then run on a mass-spec after a tryptic digest. You would need to talk to a mass-spec specialist for the last option, they will know the details and how much protein you would need.

Many times (but not always) a phosphorylated protein shifts sizes in a gel. You can try to do an IP of the endogenous protein, split the IP in half and treat one half with phosphatase. If you see a change in size between the two samples, you likely have a phosphorylated protein. It would be best if you ran Anderson gels rather than Laemmli since they tend to show protein shifts better. You can also analyze the amino acid sequence of the protein in prediction software but this only predicts potential sites. If you have purified CLC-3 protein you can also do a kinase assay using cell lysate just to see if a phosphate gets transfered to your CLC-3. Then you can run down the potential enzymes and test which one phosphorylates the protein. However, the best way has already been suggested, which is by mass spec. A very challenging and potentially expensive method but an absolute if you get positive results.

I agree, mass spec is the way to go. if you are able to get enough protein to visualize it in a coomassie, then you can cut it, and do the mass spec. You just have to keep in mind that doing a trypsin digestion there will be sites that you cannot see due to the size of the fragments you get, but you can always try another enzyme for the digestion (if you have problems with the trypsin)

Thanks for all the advice, bob1, rkay447 and laurequillo. Mass spec is probably out of my league at the moment so my best option is probably the kinase assay.

TimUCR on Thu Jul 29 18:58:22 2010 said:

TimUCR on Sat Jul 24 01:01:54 2010 said:

MS/MS as suggested is helpful if enough protein is available; for a rough estimation you may check with specific anti-phosphotyrosinyl or -serinyl or threoninyl on a Western blot

TimUCR on Sat Jul 24 01:01:54 2010 said: