Hey hi. Your problem really seems to be very odd. I would like to know on what basis you have predicted the weight of the protein.

1. Would it be the "Tag" sequence that fuses to the terminal so that the MW get bigger?

2. Other post-translational modification could happen.

3. In fact, WB method to detemined MW could have variance. Try other method like mass spectrum to sequence your sample.

hey it will be like if the end portion of your gene where the stop codon is present might be truncated causing failure in termination of the gene translation. so this might be reason for that extra 12kda in your target protein. so i will suggest you to do the sequencing of your target protein.

Approximating using software http://www.bioinformatics.org/sms2/protein_mw.html.

Thanks.

1. There is no tag.

2. Could be a problem with the start and stop translation sites. Unlikely-sequence verified. However sub cloning into a retro vector may have gone haywire. Will check .

3. Wb could be variance and/or post translational. Checked some usual candidates ( phospho/ubiquitin) with no luck. 12 kda seems a bit of a stretch?

4.will check amino acid balance.

5. Protein sequencing or mass spec may be the only option or trypsin digestion

Gael Cristofari is right. Depending on the sequence, a charged protein can show a very different migration in the electric field, even if the sequence is completely right and there are no unexpected modifications.

For more information you can read this:

Takano, E., Maki, M., et al. (1988) Pig heart calpastatin: identification of repetitive domain structures and anomalous behavior in polyacrylamide gel electrophoresis.

Do you have the possibility to do a mass spectrometry (or let it be done)? Doing so, you can check the protein sequence and if there are any degradation/oxidation products of your protein in your sample.

Good luck.

In which microorganism have you expressed the protein? In my experience the protein molecular weigh can vary from theorical calculated by cellular modifications, as glycosylation, phosphate added, etc.. But I never saw 10 kDa of difference.

I agree with the hypothesis about migration of charged molecules in SDS-PAGE, I have also seen quite large differences caused solely by this phenomenon. Also, it is worth bearing in mind what kind of markers you are using. For example, NEB prestained markers have Coomassie Blue covalently bound to them which can result in them not being the most accurate for size determination.

Thanks Mike, how large a difference have you observed? (not using prestained markers but invitrogens magic mark)