In order to produce a chimeric antibody, I transformed HEK293T cells with two plasmids: one for heavy chain and the other for light chain IgG. The supernatant was purified in a Protein G-Sepharose column and concentrated using amicon ultra centrifugal filters. The SDS-PAGE showed the correct bands for heavy and light chains, but as you can see, there are two bands for heavy chain.
Does anyone know what it means, please?
I'm curious about the background speckling on your picture. Is this a Coomassie blue stained SDS-PAGE or is this an autoradiography of labeled protein?
Back to the issue of the heavy chain doublet - I suspect that since you are using HEK cells, this might be due to the presence/absence of the normal N-linked glycosylation that is normally found on Ig heavy chain.
The easiest way to prove that would be to produce your chimeric antibody while culturing your HEK cells in the presence of tunicamycin, which inhibits N-linked glycolsylation. If this is due to differential glycosylation, the upper band should dissappear in the material prepared from tunicamycin-treated cells.
For me, it doesn't look like glycosylation heterogeneity (very sharp bands) or the presence/absence of the glycan chain (very small difference in SDS-PAGE mobility ).
It difficult to say, but it looks like some kind of signal peptide misclevage or H-chain partial fragmentation.
It also could be another heavy chain plasmid contamination.
I suggest you to do a mass spectrometry of the purified fraction and work out the two molecular weights observed, maybe you have two covalently bound chains.
Are you working under reducing conditions (DTT, BME)?
Hi, Alejandro. Yes. I am using DTT before running the SDS-PAGE.
But if it is two covalently bonded chains, shouldn't the weight be twice bigger?
Hi Priscila,
Can you provide the molecular marker you used??
In response to Dmitri Dormenshkin'd comment, the N-linked glycosylation adds ~ 3,400 daltons to the molecular mass. The secreted form tends to be rather uniform with respect to sugars.
Another possibility is that certain immunoglobulin heavy chains are covalently linked to TGF-beta. A TGF-beta monomer would add ~ 12.5 kD, which I would expect to cause a larger shift in band size than your gel shows.
Dear Alejandro, the molecular marker is added to the gel (see attached, please).
I see that the larger specie is more or less twice the molecular weight. I think that SDS are not 100% quantitative assays.
You should try doing mass spectrometry and work out which species are they, you will have the exact MW of the species. For example if your Heavy Chain is 56543 Da (invented) and the other specie is 56543*2 - 18 you may have a covalent dimer. I'm just doing some hypothesis
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