Hello
I would like to know whether my protein of interest is phosphorylated or not?
I have antibody for the protein (not phosphorylated form), but I am looking for the method to detect phosphorylated protein.
PS: No Phosphospecific antibodies are available for this protein.
Looking forward to read your comments and suggestions.
Regards,
Shirin
You can see on western blot whether protein, which is downstream from your protein of interest is upregulated or downregulated. But the tricky part is to find that downstream protein, which do not participate in signal propagation in many signal cascades. It must be quite specific. Otherwise you will se it activated, even though protein of interest is downregulated.
if you separate your Protein lysate far enough on a SDS-PAGE, you can see a band above your (hopefully) specific non-phospho band. That is your phosphorylated Protein, which is slightly bigger (because of the phospho-group), and therefore a tiny little bit above your band. If you Analyse just this bigger band you can determine changes in phosphorylation of your Protein of interest
Many thanks for the valuable suggestions, I think about RIA with p32.
What do you think about it ?
Dear Prof. Kou,
Many thanks for the valuable suggestions.
Regards,
Shirin
Phosphatase Reaction—Phosphatase reactions were taken from previous protocols (45). After anti-FLAG immunoprecipitation, the resulting washed beads were incubated with λ phosphatase (Roche Applied Science) or PP2A (gift of David Brautigan, University of Virginia) for 30 min at 30 °C in the supplied reaction buffer (λ phosphatase, in a 100-μl mix containing 100 units of enzyme in the presence or absence of the phosphatase inhibitors, 1 mM sodium pyrophosphate and 1 mM sodium orthovanadate). All reactions were stopped by the addition of 40 μl of 5× sample buffer (62 mM 2% SDS, 2% β-mercaptomethanol).
http://www.jbc.org/content/279/25/26201.long
Instad Flag antibody you can use antibody again your protein and pull down antigen/antibody complex by protein A,or G or L resin.
You can try to do a Phos-tag Western Blot and compare with a non Phos-tag acrylamide gel or something like that:
http://www.phos-tag.com/english/index.html
(use also a control since the phos-tag may cause a sift by itself)
You could use Peptide Atlas to determine which peptide has the phosphorylation site of interest (www.peptideatlas.org). You could then look for that phosphopeptide by mass spectrometry following immunoprecipitation and trypsin digestion. This is pretty standard. A simpler way is to immunoprecipitate the protein and use a pan-antiphosphotyrosine or antiphosphoserine antibody to see if you can detect a band at the right molecular weight, but anti-phosphoserine/phosphothreonine antibodies can be touchy. In the old days, we would label the cells with 32P, immunoprecipitate the protein, run a gel, dry the gel and look for the labeled band by autoradiography. This is very very sensitive.
Many Thanks Henry,
I wanted to do RIA, as you mentioned it is the old but sensitive method. My lab is concerned about radioactive contamination.
Regards,
Dear Elena, thanks for your great suggestion. I will give a try and I will write about the result here.