Hi there,

There are a lot of proteins which actually run slower/faster than expected on SDS/PAGE because mobility depends on the actual charge beared by the proteins and therefore on the weight ratio between SDS and protein. Theoritically  this ratio is considered constant for any protein (which is the basis of the migration according to MW only) it is not always the case. For instance, a protein binding slightly less SDS molecules than expected will run slower than the protein of the same MW binding SDS normally.

In your case the best thing to do is to identify the content of the amplified band (by MS fingerprint for instance).

Hi there,

There are a lot of proteins which actually run slower/faster than expected on SDS/PAGE because mobility depends on the actual charge beared by the proteins and therefore on the weight ratio between SDS and protein. Theoritically  this ratio is considered constant for any protein (which is the basis of the migration according to MW only) it is not always the case. For instance, a protein binding slightly less SDS molecules than expected will run slower than the protein of the same MW binding SDS normally.

In your case the best thing to do is to identify the content of the amplified band (by MS fingerprint for instance).

Hi Gaurav,

Since this is an in-vivo expression, I can suggest a few possibility for the nature of the  slower migrating band.   Proteins that are post-translationally (PTM)  modified tend to have slower migration during SDS-PAGE, when compared with their unmodified version.

This is because PTM add weight to the calculated molecular weight of the protein.  Eg., ubiquitin adds ~8 daltons to the protein molecular weight.  If this is the case, it is easy to confrim; during internal analysis (in-gel digestion followed by protein ID) you will identify peptides from the protein of interest as well as from ubiquitin itself.

Phosphorylated proteins also tend to have a slower migration although some proteins will have faster migration after they are phosphoryalted.

Glycosylation, methylation all alter the apparent molecular weight and mobility of the protein during SDS-PAGE.  Start by confirming that both 30 and 40kDa bands are identified as the protein that you are trying to express.  Next, see if the experimental data can be used to look for any modified peptides especially in 40kDa band.

Good luck!

Hediye.

Another possibility is read-through of the stop codon. Check the sequence 3' of the stop codon to see if there is an open reading frame of the right size. Read-through can be reduced by adding a second stop codon.

In my experience, higher apparent MW on SDS-PAGE is quite common for proteins with high Glu/Asp content... See for example:

Graceffa, P., Jancsó, A., & Mabuchi, K. (1992). Modification of acidic residues normalizes sodium dodecyl sulfate-polyacrylamide gel electrophoresis of caldesmon and other proteins that migrate anomalously. Archives of biochemistry and biophysics, 297(1), 46-51.

Alves, V. S., Pimenta, D. C., Sattlegger, E., & Castilho, B. A. (2004). Biophysical characterization of Gir2, a highly acidic protein of Saccharomyces cerevisiae with anomalous electrophoretic behavior. Biochemical and biophysical research communications, 314(1), 229-234

Thank you all for your valuable feedback. 

Certain serine proteases non-colavently complexed with inhibitors survive boiling in SDS.  Classic examples are plasminogen activators complexed 1:1 with plasminogen activator inhibitor type-1 (PA*PAI-1).  This does not readily explain your higher MW when expressing a single protein, but it demonstrates that protein-protein binding is not universally disrupted by ionic detergents.

This is a common phenomenon. Many proteins migrate at different apparent mw to expected.  Since this is an E.coli expression, the effects of post translational modification are unlikely to cause a 10 kDa shift in migration. The most likely reason is that your protein has an overall positive charge at the pH of the gel and/or loading buffers even when coated with SDS. Check the pI of your protein and work out the overall charge it may have at the pH of the gel/buffers. 

Normally, aberrant migration is not a problem. In the first instance, carry out a mass-spec on the band that appears to be your protein. If that shows the correct molecular mass then you're on the right track! After that, you'll be able to confidently identify your protein on SDS PAGE.

hey gaurav. did you get any solution for this problem. i am also facing the same problem.if u have found plz share

I am handling serine rich protein with high lysine content. I am facing the same problem that my protein shows high molecular weight . Can you explain this please?