Run Western blot against the his-tag

Omar Gonzalez-Ortega I did and I got multiple bands. Not sure if it's because I used more of primary and secondary antibodies. I used Abcam's Mouse Anti His Monoclonal antibody (1/1000) and Goat anti mouse pAb as the secondary(1/3000), as recommended by Abcam. Please see the western blot image. Clones 1,2,3 are different colonies I tried.

Try using an antibody against your protein of interest..

Sometimes protein molecular weight estimates from protparam doesn't always run at that size as proteins can undergo posttranslatiinal modifications such as glycosylation etc.

Bubacarr G Kaira Unfortunately, I don't have antibodies against my protein!

Expected band size is 65kDa, where some researchers have got the same protein at 75kDa(could be glycosylation). But am confused about the lower molecular weight bands and it's hard to identify if my protein is expressed or not.

Well there is a lot of nonspecific interaction. Your kit does not work.

@Anju. The lower molecular weight bands could be degradation products from the higher molecular bands.

Mass spectrometry of the 75 kDa band will tell you whether it is you protein of interest or not..

Do you have an X33 without any insertion you can use as a negative control?

Pablo Pérez-García The negative control I used has empty plasmid transformed into X33. I haven't used X-33 without any insertion.