How about a dimer? There are several reports that people see protein oligomers in SDS-PAGE. You may try more harsh buffer conditions to separate the dimer, sometimes also cooking of the sample may help.

How about a dimer? There are several reports that people see protein oligomers in SDS-PAGE. You may try more harsh buffer conditions to separate the dimer, sometimes also cooking of the sample may help.

Thanks a lot.

Yes， May be their dimer. I cooked this sample 30 mins and added high concentration SDS. It shows very strange bands compare with control (GFP-his).  

This protein is the C-terminal of a large plant protein (84KD), which is also around 130KD  in the Western bolting. Is it possible to be lipid binding modification? 

I have worked with a protein that form stable dimers that can't be denatured by SDS. It often give two bands even after cooking.So I will suggest that you should try denaturing with 8M urea before loading a small amount of it on SDS PAGE, as 8M Urea tends to give intense bands.

Is this gel reducing or non-reducing?  When running gels it should be a habit to run reduced and non-reduced samples on the same gel.  If you are very certain with the MS data showing monomeric mass and it's not the +2 ion then the protein might be forming a covalent dimer in situ upon SDS-PAGE analysis.  If you did LC/MS then there was one peak on the LC?  Does this protein have cysteines and if so are there an even or odd number?  The LC/MS and SDS-PAGE were conducted on the same day?  

Some SDS-PAGE considerations:  IgG1 run reduced and GDF15 run reduced will partially re-oxidize in the gel as fresh and sufficient reducing agent migrates away with IgG1 forming some double heavy chain, some double light chain, and some heavy-light chain while GDF 15 will partially reform intra-disulfide bridges.  This will not happen if reducing agent is in the cathode chamber(NuPage gels) or the sample is also alkylated once reduced.  If run oxidized then inter-chain disulfide can form during the heating step or in the gel unless there's alkylating agent present , NEM or IAA.  With non-reduced samples this oxidation can happen with proteins having an unpaired cysteine like GCSF and even with even-numbered cysteines disulfide bridged but with a trace of reducing agent present (disulfide exchange) at the heating step.  Under extreme conditions a cysteine can desulfurylate to form beta alanine and then a lysine's amine side chain adds across the double bond to create a N-C bond, but this is unlikely.   

Bacteria do have lipoproteins usually in the inner membrane of the periplasm for virulence and fortunately for host immune responses.  In E coli it is a very unusual structure in which the N-terminus is a cysteine that has a glyceryl group on the S and N atoms and the glyceryl group is tri-acylated.  Would you expect your plant protein to be recognized by this E. coli pathway?  If you think lipid is present then the gel can be stained for lipid content.  If the LC/MS is picking up a 24kDa lipoprotein monomer then the lipid would have to dramatically alter the charge/mass ratio and Stokes radius on the SDS-PAGE to account for 2x the MS mass.

Thanks a lot. I will try for 8M urea.

it is  reducing gel. There are no cysteine in my protein

Not having cysteines in the sequence is basic information that helps from the very beginning.  If you are absolutely certain about the mass spec data then this makes me suspicious of another phenomenon that occurs in SDS-PAGE.  There's a reason why you asked about a possible lipid modification that could happen in the E. coli expression.  Does this protein have any relationship to membranes/lipids?  There are some examples of proteins that have unusual hydrophobic amino acid content and conformation, certain transmembrane domains or oxido-reductases for example, that become aggregated in the presence of SDS and is even more favorable when heated. There may even be micelles formed with SDS.  Without further explanation, what happens when you do not heat the samples for SDS-PAGE and what results do you see with SEC analysis?

I have had experience with a 25 kDa protein (theoretical) that ran at 50 kDa on SDS-PAGE. It was due to the low complexity of the primary amino acid sequence which meant that it did not bind SDS efficiently, thus a spurious MW by SDS-PAGE. This protein also ran at over 100kDa on size exclusion chromatography as it was intrinisically unstructured. You should look at your primary amino acid sequence for amino acid bias.

Thanks a lot.