Did you check the authenticity your protein running on 10kDa by mass spec? may be the running behavior of your protein need not to be the one running in the control protein (in the ladder that shows 15kDa)?  Western blot with monoclonal Ab may also a good start to prove!

Did you check the authenticity your protein running on 10kDa by mass spec? may be the running behavior of your protein need not to be the one running in the control protein (in the ladder that shows 15kDa)?  Western blot with monoclonal Ab may also a good start to prove!

Hi! Hard to tell, MS and blotting are good starting points as mentioned above. Maybe some posttranslational modifications (eg glycosylations) play a role?

To AAJ Vasudevan: The MALDI also gave me a weight of 10,700. I think that the protein itself is not the one I want and am wondering if this may be due to a phage contamination to my E. coli stock. Ab's are a good idea though, thanks for your help and I will look into that.

to V Weiss: Possibly, but the protein is smaller than what I am expecting. Do you know what post translation modifications would be able to cleave a protein? Thanks for your help.

Hi! Glycosylations influence the number of SDS molecules that can attach to your analyte and therefore the MW appears higher when your protein is glycosylated (less SDS attached). For MW determinations on the Bioanalyzer this deviation is really impressive, but you can detect the same effect also with SDS PAGE. You can have a look on an application note for Agilent prepared by a colleague of mine:

5991-3435EN.pdf

Have you tried to digest your protein and identify it via its fragments (MALDI MS)?

run another protein of similar size like lysozyme 14 kda or cyctochrome c 10 10 kda along with ur purified protein

Hi, 

I think you can exclude glycosilation. The process is quite specific. We are trying to express a glycosilated protein and it is not an easy process.

If you have a MALDI instrument, why don't you do the following?

Digest an aliquot of your protein with the appropriate enzyme (based on the sequence you expressed) and run the digest with the MALDI.

A simple peptide fingerprint DB search should give you enough information to understand where the problem is.

Hello,

Sometimes it happens in SDS PAGE that protein gives different size not  coinciding with the theoretical molecular weight of the protein,But it doesn't mean that protein is not the right one. For the sack of science it needs to be given next route of purification to be analysed by MALDI.

For Example same happened with my research my protein was supposed to give 32 KDa but it was appearing at 37 kDa On SDS PAGE. But it was perceptively active.

good luck.