Good time of day!

I am trying to determine whether my protein is a multimer or a monomer by NativePage.

It is expressed in e. coli as an MBP tagged version, with the option of keeping or cleaving off the tag.

It is suspected that the protein is a dimer since it consistently fails to separate from cleaved MBP on a size exclusion chromatography column (as a monomer it is about 25 kDa, so about 50 kDa as a dimer, and it comes out as an overlapping peak slightly earlier than the 43 kDa MBP).

I ran several samples on a 4-16% NativePage Novex Bis-Tris gel.

On the gel, lane 1: MBP-tagged version of the protein;

lane 2: the protein alone

lane 3: MBP alone

lane 4: a mix of the protein and MBP (cleaved off)

The green box shows the MBP position, the blue and orange boxes show the approximative theoretical MW of the monomer and dimer respectively (+/-15% to account for size estimation error mentioned in the NativePage Novex Bis-Tris gel system protocol).

Off the bat, the MBP appears to be heavier than the expected 43 kDa, despite being a globular protein and having an acidic pI (so theoretically migrating relatively fast). It also comes out as two bands (three if more protein is loaded) - which I am assuming is due slight conformation differences? (should not be due to gel quality, it is new and was stored at 4°C).

So already, the first question is, why does MBP appear heavier?

The protein is even stranger, as it appears much heavier than a dimer (related proteins can be monomers up to tetramers, but are usually dimers). There is no obvious reason for it to be migrating much slower (it has a pI similar to MBP, it's a soluble protein, not a glycoprotein and there shouldn't be any glycosylation). My only explanation besides it being a higher multimer, is it possibly having a shape that slows down migration (no structures available in the literature, so can't confirm). But can protein shape alone slow down migration that much?

There is some salt in the protein alone and protein+MBP mix samples, but below 50 mM (as required per protocol), but no salt in the MBP-protein or MBP samples (I've read this can affect migration).

Alternatively, if it is a higher multimer, then why doesn't it separate from the MBP by size exclusion chromatography?

Has anyone else had problems of proteins seeming inexplicably much heavier on a Native gel ?