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Detection of phosphorylation site


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#1 TimUCR

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Posted 23 July 2010 - 05:01 PM

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

#2 bob1

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Posted 25 July 2010 - 03:14 PM

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.

#3 rkay447

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Posted 27 July 2010 - 09:21 AM

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.

#4 laurequillo

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Posted 27 July 2010 - 10:23 AM

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)
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#5 TimUCR

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Posted 29 July 2010 - 10:58 AM

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.

Edited by TimUCR, 29 July 2010 - 11:13 AM.


#6 laurequillo

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Posted 29 July 2010 - 12:56 PM

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.


You can try to find a lab with the machine, so you dont have to pay, just give them a colaboration.
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#7 kottila

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Posted 04 August 2010 - 12:06 AM

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


Put in radioactive P, treat your sample in a way that will phosphorylate your protein and then run it on a 2-d gel. If you know where your protein is situated, just cut it out and send it to a mass spec lab.

#8 Inmost sun

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Posted 04 August 2010 - 11:00 AM

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

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



#9 laurequillo

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Posted 04 August 2010 - 10:14 PM

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


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



Yeah,you can Ip your protein and then do a wb using those Ab.

If you can see an upshift in your proteinyou can use as well lambda phosphatase to check if it is reallyphosphorylation. But those methods wont give you sites, just general information
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#10 TimUCR

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Posted 10 August 2010 - 11:26 AM

Thanks for the suggestions everyone. There aren't phospho-specific antibodies for chloride channels as far as I can tell. I would prefer to just see if it can even get phosphorylated before I send it off to a mass spec lab. Instead of doing the kinase assay with the phosphate isotope, could I do a gel shift assay on SDS-PAGE instead? If the protein gets phosphorylated it should run slower? Will this be significantly noticeable or would it just be easier to do the kinase assay?

#11 laurequillo

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Posted 10 August 2010 - 11:38 AM

Thanks for the suggestions everyone. There aren't phospho-specific antibodies for chloride channels as far as I can tell. I would prefer to just see if it can even get phosphorylated before I send it off to a mass spec lab. Instead of doing the kinase assay with the phosphate isotope, could I do a gel shift assay on SDS-PAGE instead? If the protein gets phosphorylated it should run slower? Will this be significantly noticeable or would it just be easier to do the kinase assay?


If the protein runs slower (not always the case...) then you can treat your sample w/o phosphatase and check that what you are seeing is really phosphorylation (and not sumoylation, for example...)
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#12 TimUCR

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Posted 10 August 2010 - 01:02 PM

Is it likely that there won't be any mobility shift?

#13 mdfenko

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Posted 11 August 2010 - 11:31 AM

i used to do gel shift on urea-page (no sds or reducing agent). my protein was 20 kDa.
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#14 laurequillo

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Posted 11 August 2010 - 11:35 AM

Is it likely that there won't be any mobility shift?


It depends on your protein and the phosphorylation. I guess the up shift depends on the number of sites, and the percentage of protein phosphorylated.
Sometimes I saw some proteins phosphorylated by protein X in one site with and upshift and by protein Y in another site and no upshift.

Edited by laurequillo, 11 August 2010 - 11:36 AM.

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#15 mdfenko

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Posted 11 August 2010 - 11:37 AM

with urea-page we saw a downshift of the phosphorylated protein (due to increased charge).
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