lots of www site will give you infos about what PTM has been found

as a starting point

http://www.phosphosite.org/homeAction.do;jsessionid=82C167AAD91E6EB986E3E536B317A5E7

http://www.biosino.org/SysPTM/index.jsp

1. Look into the literature first what is known about regulation of affinity (i suppose here we are talking about afinity towards another protein) by post translational modification.

Some of the handy methods (available in almost all labs)

1. e.g. if it is phosphorylation: phosphospecific antibodies for your protein of interest , if available, you could use to directly check phosphorylation.

Otherwise go look for : antibodies to detect the phosphorylation of specific amino acids (pS, pT, pY). Immunoprecipitate your protein of interst and look whether you you get a band with any of these.

2. You could check whether Ubiquitination is involved: Immunoprecipitate your protein of interst and look whether you get a band with polyub antibody.

sometimes simply higher exposure of your autorad from western where you ran lysates containing your protein of interst could give you HMW bands, if UB is there.

If you dont want to be exploratory:

1. 2DGE is an option: separates proteins on the basis of charge and mw.has sufficient resolution to separate the modification states of a protein . It is often better to enrich your protein of interest( immunoprecipitation or chromatographic purifications ) to make it less complex.

2. MS is another option you have.

Gene Cards.....

Treat sample with phosphatase and re-run sample....

I suggest to take a look to this site :

http://www.nextprot.org/

Then if anything is reported you can try different experiment:

treating the cells with thapsigargin (for example) will give you phospohrylated proteins!

Hi,

Is this forum still active?

Could someone tell how much protein is needed to study PTM through mass-spectrometry? I will use a purified recombinant protein.

Thanks

The answer is so easy: by the Mass Spectrometry.