I have run a 2D gel to identify certain protein isoforms in rat urine. I did a western with a antibody that binds to all isoforms of the protein. The expected size is around 17kD-20kD., however the antibody shows spots that are around 30kD and 60-70kD. These spots have the same pI as one of the isoform found in the appropriate range (17-20kD) so i think it is a PTM on that isoform.
My question is what kind of PTM can cause this huge change in molecular weight but not change the pI of the isoform? i thought it could be glycosylation but I assume that it would cause a change in pI
Hi -
Sumoylation could be a probable candidate for what you are looking at. Most organisms seem to have SUMOs which are around 12-15 kDa and if you add that up with your Mw, you get your ~30kDa spot. The higher 60-70 Kda might be background - although SUMO attachments are known to also occur as conjugates. So that could be a possibility? Maybe? You can try digging into this a bit more. As far as I know SUMO modifications have a pI of about 5. Trying running the protein sequence through a software called SUMOGps or Abgent's SUMOplot. That should tell you whether your protein has the possibility of getting sumoylated.
Could it be dimers of your protein? Seeing that the weight seem to have more or less doubled. Sometimes not all binding gets destroyed during denaturation, so you can have dimers appear on your gel. However, i have not much experience with 2D gels. But this is something which is see some times appear in my regular western blots.
Covalent cross-linking by transglutaminase is also an option. Predicted MWs are based on the primary sequence, but there are many proteins that show a different relative MW, simply because they bind SDS differently or have secondary structure that alters their electrophoretic mobility, e.g. MHC-class II molecules form SDS stable dimers. Remember the pI will stay the same as long as the net change is equal, i.e. a +ve for each -ve equals a net change of 0.
Hi! As stated before, maybe you detect oligomers. Another option causing your problem might be crossreactivity of your ab. So maybe it would be good to extract your spots and then do some protein identification via MALDI.
Hi,
I agree with the oligomerization and crossreactivity. Otherwise amino acid composition might change also the SDS-PAGE molecular weight. E.g. RNaseE 118/180 kDa.
In you case, oligomerization or crossreactivty are more likely.
best
I would start by theoretically assessing the pI of your protein (probably know this already), and compare with theoretical pI of modifying proteins like SUMO as suggested above. If same pI, then you will expect spots with different Mw but same pI.
Non-complete focussing of the first dimension can also blur results and I have experienced with 2D gels that a pool of my enzyme have appeared as a ladder with same pI, while a pool has focussed nicely with various pI range dependent on the modification on my protein. Reducing the amount of protein loaded on the strip has solved this.
I agree with dear Lise Bjørkhaug in reducing the amount of protein loaded on the strip. Also in my opnion it might be dimer for bonds in near to 30 kDa, and be Albumin for bonds around to 60 - 70 kDa, in case albuminori has happened in the sorce urine! May be if you change the source of urine you will got different results.
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