What post-translational modifications help proteins migrate faster on an SDS-PAG - (Apr/16/2015 )
My HA-tagged protein A appears to have two more bands when another FLAG-tagged protein B is co-transfected(as opposed to a single band when A is transfected alone). The two new bands run right below the original band and I assumed the original band is post-translationally modified. One of the two new bands is only a little less abundant than the original band, whereas the other band is significantly weak. I was just wondering what kind of post-translational modifications would let the protein migrate faster. Thank you.
phosphorylation might cause the sample to run faster.
mdfenko on Fri Apr 17 12:18:47 2015 said:
phosphorylation might cause the sample to run faster.
Thanks for your reply mdfenko, I also saw people mentioning that but did not find a example. Do you have example of that?
Hi,
is FLAG-tagged protein B an ezyme or could interact with HA-protein A expression pathway?
pavoni.ernesto on Fri Apr 17 13:04:44 2015 said:
Hi,
is FLAG-tagged protein B an ezyme or could interact with HA-protein A expression pathway?
Hi Thanks for your reply.
As far as I know B has no enzymatic activity. However, B does physically interact with A. B could also transcriptionally activates endogenous A, which is not detectable by anti-HA.
Hi,
another question, your endogenous protein A undergoes a process of maturation?
example: pro-Protein A cleaved to Protein A
mdfenko on Fri Apr 17 12:18:47 2015 said:
phosphorylation might cause the sample to run faster.
I find this really interesting. I've never seen this and am also curious about proteins that could migrate faster when phosphorylated. I know they exist from anecdotes and a few papers I've run across. It's curious behavior, because except under exceptionally acidic conditions, the phosphates should impart additional negative charge and ostensibly increase the force in an electric field. Yet this doesn't seem to be the dominant factor.
I think that a phosphorylation event could result in faster, slower or no change on SDS-PAGE. It all depends on the protein and running conditions; its charge/isoelectric point, the composition of the amino acid sequence around the phospho site, and its interaction with SDS and the gel matrix will all influence its migration.
I look at several phospho proteins in the lab and they all run larger. So the mobility appears to be more strongly dictated by forces other than electrical charge (at least for the proteins I look at). I'm partial to the possibility that SDS interacts less with the region of the protein that is phosphorylated. This could change its shape and behavior in a gel matrix.
I would still expect that the acidic/alkaline nature of a protein and the choice of buffer/pH would have some influence on mobility in response to phosphorylation, but this may be masked by physical changes in the protein as suggested above. The pKa of dianionic phosphate ester -O groups is low (~2 and ~6). So depending on the buffer you use and the resultant pH during your gel run, you'll probably be operating with dianionic phosphates (except at lower pH ranges, i.e. MES, some Bis-Tris formulations). Depending on the net charge of your protein at the given pH, and the number of phosphates, you could estimate the impact of phosphorylation on the overall charge during electrophoresis.
I'm sure this is all explained nicely somewhere within the JSTOR archives 
(edited for typo)
In grad school I worked on a protein in which 90% ran at the expected molecular weight but ~10% ran 5kDa faster.
Initially we thought it was a proteolytically degraded form of the protein, until MS data showed it was full length protein.
Long story shot...turns out the faster migrating band had a covalent intra-molecular crosslink.
^That's really interesting. What was the nature of the covalent crosslink, out of curiosity? I'm assuming this wouldn't have been an easily-reduced disulfide if it took mass spec to reveal the chemical basis of your observation.