I tried to purify a beta propeller domain fused to MBP protein from BL21. I got a quite decent amount of protein, however, it doesn't run at the expected size on SDS-PAGE gel. The expected size is 77kd, and it ran as a doublet(similar amount) at around 50kd on the gel. Does anyone have ideas about this problem?
Hi Lan,
Migration of the protein is can be influenced by the charge, mass and the charge of the molecule. It could very well be that the protein is folded in such a way that it does not migrate as you would expect, for example it might be folded in a tight ball and migrate faster - therefore appearing smaller.
Then, it may also be that there is some degradation of your protein. Are there any other bands or smears visible?
Kind regards,
Chris
Hi Lan,
Out of interest, what loading buffer are you using & which gel conditions? Sometimes if the reducing agent is either losing activity or insufficiently potent for the protein involved you can get proteins that also run at unusual lengths, or form oligomers etc, and preparing a new batch of the same buffer (i.e. beta-mercaptoethanol Laemmli buffer) is needed to get properly medieval with the protein in question. In my hands, the DTT version of Laemmli is also less effective at denaturing the protein than the B-ME, so I default to the latter where possible. As Chris says above, some proteins just don't run as expected based on folding if they are very stable, or if they possess highly charged regions that behave unusually in electrophoretic conditions.
Hi Lan,
probably you have done it already, but I would like to remind to check that the buffer of your chosen marker and your protein loading buffer are similar to ensure the same running behavior of marker and protein (because of salt etc) and that you sequence your expression vector for premature stop codons. As Chris J Visser, I also think that your duplex originate from protein degradation either in E. coli or during the purification. There are a lot of opimization protocols available online. Also, a duplex could arisefrom auto-phosphorylation (in case of kinases) or seldom from strong DNA or RNA binding (even in SDS). But that is probably not the case for your beta propeller protein.
good luck
Hi all,
Thanks for the suggestions. As to the buffer and ladder I used, I've been using the same for a while and it works fine for all other proteins. I used fresh DTT in my loading bf and I boiled for 5min. Here is my gel picture. Do you think there's some degradation going on? I have included PI cocktail and PMSF during purificaiton, but I'm not sure if there's degradation going on in BL21. I'll check that today.
Hi Lan,
thats a fine gel and a really good purification you did. I think the bands below your duplex are degradation products, but in low amount compare to the duplex. Sometimes some degradation even with cocktail and PMSF can not be avoided.
I would not be so concern about the size difference of your duplex to the estimated size. You can verify your protein by the sequencing data and a Western blot with MPB antibodies. Maybe you can check the isoelectric point of your protein. In case of an extrem low pI you may have an explanation for a different migration behavior.
I cannot explain the duplex. It seems like modification (phosphorylation, but it is unusual in E. coli). If you have to really know and excess to MS or MS-MS at your university, go for it. Your can verfiy your protein once more and know by the exact difference of the sizes what kind of 'modification' it is.
Hi Lan,
Did you dialyzed your protein after purificatin?
Maybe if the dialyzed protocol is too long( for example over night), you protein could be degraded by proteases. This is occured in my recent experiments! I checked my purified and not purified protein by LC-MS to be sure about the MW.
Hi Lan,
Did you check the pI of your protein? Some proteins with too many charges do not bind SDS as they are supposed to do, hence weird positions on SDS-PAGE. I agree with Concetta and Michael, MS is the way to know if the duplex bands are your full-length protein. Western blot in this case will not tell the difference between full-length fusion protein and degradation products (I assume MBP is N-terminal fusion?) Just curious, there is a third band below the duplex, is that MBP?
In my experience, this situation is not unusual with membrane-associated proteins. There is an explanation for this in Rath et al, (PNAS, 106(6) p. 1760-1765, 2009). As far as the processing of your protein is concerned, I'm not familiar with E. coli as an expression system, but in S. cerevisiae this can be produced by internal proteases and, thus, it is not possible to avoid this problem by adding proteases inhibitors during the purification procedure. In the latter system, the use of mutants devoid of vacuolar proteases can help, but I don't know if there are this kind of mutants in E. coli
- Badges
- Science method