Some possibilities:

(1) The upper band is intact protein and the lower band is due to proteolytic cleavage.

(2) The protein undergoes post-translational modification, such as phosphorylation or glycosylation, resulting in differential mobilities on SDS-PAGE.

(3) Your antibody is insufficiently specific and is detecting another protein, too.

Dear Ihtesham

Proteins sometimes migrate differently on SDS-PAGE to how one might expect.  If your protein is not perfectly globular then it could alter its migration speed.  So, if you have two bands lighting up in your western, I expect that one is the intact ISG15 and the lower one is a cleavage product of the same.  That would be my best guess without seeing your data.

Hope this helps

Dave

Some possibilities:

(1) The upper band is intact protein and the lower band is due to proteolytic cleavage.

(2) The protein undergoes post-translational modification, such as phosphorylation or glycosylation, resulting in differential mobilities on SDS-PAGE.

(3) Your antibody is insufficiently specific and is detecting another protein, too.

I agree with David and Adam, Slightly higher band than expected in western blot could possibly be because of post-translational modification of your protein. Does your protein have any post-translational modification sites? The lower band is most probably the cleavage product of intact protein. I don't know what is the difference in the size of your expected band and these observed bands, so these will be the best guess.

Hi! I agree with most what has been mentioned before and would like to stress that some proteins run much different on SDS-PAGE than you expect from their seize. Many membrane proteins for instance bind a lot of SDS and therefore run slower.

I have also worked with a (membrane) protein that ran as a higher band when it had its substrate bound and as a lower band when it was substrate free or as two bands when only part had substrate bound.

Dear all Thanks, attached the picture, the mareker is for 15 KDa and my protein is also 15KDa, but the two band non of them is 15KDA, which one is ISG15 and why? but my questionis, same protein run differently on SDS-PAGE, but if you get two band simultaneously how will consider, which one my require protein, what will be the basis of my selection? if i consider bluntly the upper one my band then i have zero data, but if consider the lower one my protein then the data is according to my desire results, but i am confuse that why i hould consider the lower one not the upper band?

Can you cut out your bands and send them for Mass Spec analysis? This way you will know if any of these two bands is your protein and if so which one.

Another option is to tag your protein and analyse via Western Blot with antibodies where your protein runs.You could tag your protein either N-terminally or C-terminally or even better make two constructs, one with an N-terminal and one with a C-terminal tag, to look at degradation products / cleavage.

You could immunoprecipitate or immunoaffinity purify the protein of interest using your antibody and submit both bands for N-terminal sequencing.

I  do agree with all the above said answers. Are you using mini gel or big-size. Also what is the percentage of your SDS-PAGE gel? Is your Antibody is purified?