Thanks Mohammad....However the MALDI-TOF will only identify my protein after!! I identify it via the western or similar. My question is how to identify which is the DIFFERENTIALLY regulated protein in the first place!!

Any idea?

Hi! Identification of an unknown protein can be a tough project. As first shot, I would suggest a fractionation of proteins on FPLC e.g. ion-exchange. Then you can test all fractions and see if you are able to recover the activity in specific fractions.

This should reduce the overall complexity of your sample. 

Subsequently you can compare the protein pattern of the identified fraction (treated vs untreated) via SDS-Page and analyze single bands via MS. Or you can directly do differential proteomics of the fractions. 

If the complexity of the sample is still too high you can do a second FPLC run with more specified conditions, using only the identified fraction. 

But it isn´t always as easy as it sounds. The most important point is a robust screening method since every FPLC run will dilute your protein of interest. If your activity is high enough you hopefully have good chances.

Good luck!

Hi ,

I suggest to you performing MS approach.  You could just identify what ou are looking for but moreover if you are interested into observing differences upon treatment you may perform a SILAC approach.