Hi Fayezeh Shamsi SDS-PAGE is not an accurate measure of molecular weight. It is an estimate comparing a band to molecular weight standard proteins. Depending on the vendor, the MW of standard proteins can differ quite a bit from one to teh next.

Send some of your purified protein for mass determination to a mass spec lab to make sure it has the expected mass.

FLAG/Hexahis are ~1kDa/ea. so you're 2-3kDa from standard? Seems reasonable, did you have good linearity in your gel (straight lines, no bending or warping)? PAGE migrates proteins based on their denatured hydrodynamic radius, your protein may just unfold in such a way as to cause higher than normal 'drag'. Also, is this a glycoprotein or are some other PTMs expected?

Dr. Schmitz makes an excellent point. PTMS could be a reason that the protein is a little higher than expected. Did you check to see if your protein is bio-active? If your protein requires a PTM to be functional (i.e. a kinase to phosphorylate) and its functional after purification, this can be an indication that the PTM is present.

Vincent Paul Schmitz Thanks for your reply, Rdi1 is a small soluble protein and it has no PTM. I purified this protein with 6x His and there were always a clear band in 24kDa. but after double tagging it with Flag and His I saw a band around 36 kDa instead of 31.8kDa.