I would not immunoprecipitate with a phosphoserine antibody. As you said, you will end up with a lot of proteins that you are not interested in which happen to be phosphorylated on a serine. Moreoever, you would lose your protein of interest which is unphosphorylated, which you can use as a control or as a means to quantify your phosphorylation levels.

So, if you have any antibodies specific to your protein of interest, I would use those instead. Figure out their epitopes to determine which antibody to use. Epitopes that cover your phosphorylation site (or other potential phosphorylation sites) are not the best targets. If your antibody binds on a region that is not phosphorylated you can safely use that to concentrate your protein. Stain with your phospho-antibody on a western blot as well as with a STAT5 antibody to make sure your protein is present.

You will notice that you get a lot of aspecific binding. Immunoprecipitations are never 100% specific. You will get albumin and IgG bands and probably a lot of other junk that sticks to your protein. Make sure to add good controls like pure protein of interest, negative controls, etc.

Good luck!

The answer is, "it depends". Often a core antibody will also immunoprecipitate the phosphorylated form, but you will have to check for your particular antibody. Can you find the epitope for the anti-Stat5 antibody? Is it near a phosphorylated site, so that you might be concerned about steric hindrance between the phosphate group and the antibody? To test your antibody, you could immunoprecipitate stat5, and then perform a Stat5 western blot on the pellet and the supernate. If the antibody immunoprecipitated all the forms, you should only detect stat5 in the lanes from the pellet.

I would not immunoprecipitate with a phosphoserine antibody. As you said, you will end up with a lot of proteins that you are not interested in which happen to be phosphorylated on a serine. Moreoever, you would lose your protein of interest which is unphosphorylated, which you can use as a control or as a means to quantify your phosphorylation levels.

So, if you have any antibodies specific to your protein of interest, I would use those instead. Figure out their epitopes to determine which antibody to use. Epitopes that cover your phosphorylation site (or other potential phosphorylation sites) are not the best targets. If your antibody binds on a region that is not phosphorylated you can safely use that to concentrate your protein. Stain with your phospho-antibody on a western blot as well as with a STAT5 antibody to make sure your protein is present.

You will notice that you get a lot of aspecific binding. Immunoprecipitations are never 100% specific. You will get albumin and IgG bands and probably a lot of other junk that sticks to your protein. Make sure to add good controls like pure protein of interest, negative controls, etc.

Good luck!

Instead you may try PhosTag phosphoaffinity based SDS PAGE and do your blot with your total STAT5 antibody. No IP, no phosphoserine. You may want to have a look at the protocol:

http://www.wako-chem.co.jp/english/labchem/product/life/Phos-tag/

I will IP with total STAT5, which should pull down all states of STAT5! Plus you are enriching your blot with STAT5, which in the other case you will have loads of noise from the other serine phosphoylated proteins. Hope that helps

Hi Pramod,

I think there is at least 2 different way to detect your phosphorylation on Stat5: The easiest way is if you have Phospho specific a.b such as ( pSer-726) which can be used in WB. The other way is as you say pan STAT-5 Ip and Phospho-Ser WB (First if you are going to use the same membrane) and then STAT-5 WB.

Just for information, you can also use (proximity ligation assay) PLA to detect the phosphorylation. You can find some information on OLINK homepage.

Good luck

Hi Pramod,

Your anti-STAT5 Ab will precipitate both forms of STAT5 as long as epitope is not located on phospho-Ser site. Your are right you can identify phosphoyilated form of STAT5 with anti-Ser Ab then

Amina.

Hi Pramod,

1. If you IP with phosphor-Ser antibody, then you just have to use total STAT5 Ab to show Stat5 is IP'ed. However, total phosphor-ser Abs are not really your best tool for the first step since they are not really that good to begin with in total lysates.

2. So the better way is to IP all the STAT5 and then blot with anti-Phospo STAT5 Ab. NOTE anti-phopsho STAT5 .... You could also use the total phosphor-serine Ab at this step to detect Ser-P on the IP'ed STAT5. This signal will much stronger than (1)., and more specific. If you use anti-P STAT5 Ab, you can look at specific sites of phosphorylation. This will give you the best overall signal, and specificity.

3. OR Like Faud said, you could just directly blot a total lysate with anti-phosphoSTAT5 Ab. This is the easiest. But your signal will be lower. If lysate is limiting, then IP is better.

Good luck !

thank you all your great explanations... I will plan my experiment according to your suggestions and advice.

Unlike the anti-P-Tyr monoclonal Abs, anti-P-Ser Abs often do not work very well. There are actually panels of Anti-P-Ser that you can try, they are made against P-S in different contexts (Cell Signaling Technology). Even those do not recognize all P-S proteins. But, in general IP your protein first with the specific Abs and then blot for the P-ser. You may also find an antibody specifically made against P-STAT5, which would be a better reagent than anti-P-Ser.