My protein of interest is actually of 39kDa and after purification I am getting at 66kDa and through MS/MS analysis it is proven that the band is actually the same 39Kda protein. My lysis buffer includes 50mM TRIS, 200mM NaCl, 10% glycerol, 1mM PMSF, 0.1% triton X. After purification when I am dialysing or concentrating (with sucrose and centricon) it get degraded. Can anyone please suggest me the reason and solution?
The first lane near marker is of protein just after concentrating it and the next lane is empty and the lane after that is of protein just after dialysis..
Dialysis buffer is 50mM Tris pH 7.8
Those gels you showed, are they reducing or non-reducing SDS-PAGE? Does your protein have cys residues? Have you done Western blotting to ensure that those bands appearing after concentration are degradation products? Is your protein a protease?
No Martin my protein is not protease and the SDS gel is in reducing state with DTT added in the SDS loading buffer. Yeah protein do have Cys residue. And i haven't done western to ensure the degraded products.
I'm a bit confused. The first gel appears to show a very nice purification of a roughly 66k protein, assuming the lanes on the right are raw lysate and the lanes toward the left are purifications.
...but then the second gel appears to simply show two raw lysate fractions. If your protein is in there somewhere (I have no idea where, or how you'd tell, given all the other proteins), it's probably getting degraded by one or several proteases floating in the lysate, probably because you're dialysing away all your protease inhibitors.
If the second gel actually shows two protein purifications....then that's not very good protein purification. Get your protein purified to the extent shown in the first get (so you have a single clear band on a gel, plus some very faint other stuff), THEN concentrate it and dialyse it. And maintain protease inhbitor concentration wherever possible. Also, possibly consider using something more comprehensive than just PMSF (Roche complete tablets are pretty good).
Hi Dhaval Patel, if the second gel is the same sample of the first gel (purification) just after concentration, it is clear that your protein is not pure after your purification protocol, and the degradation that you see if because a contamination of a protease. In fact, as you can see, there are anothers bands presents in the lines corresponding to your purification fractions. Try with more protease inhibitors at least in the first purifications steps.
Thanks for your reply John and Hector, actually the second gel is of same sample shown in first gel but its after concetration and dialysis so there are so many bands which i assume that protein is degraded.
Also, knowing what kind of purification you're doing would help. Given that it's in the HIS tag topic, I'll guess HIS tag, but this isn't explicitly stated. Also, how is your protein expressed? I'm assuming E.coli, but again this isn't stated. If so, Is it happily soluble in the cell or does it form inclusion bodies?
Generally speaking, you shouldn't need to concentrate up raw lysate before column purification, and in most cases doing this will be fairly counterproductive. Not only are you concentrating your protease activity, you're also increasing the chance of things spontaneously precipitating and/or clagging up your column.
Nickel affinity purification should be more than capable of pulling your protein of interest out of even fairly dilute lysate (you can always run it through the column several times if you're worried), and this will get rid of an awful lot of protease activity (as the majority of the proteases, as well as most other proteins, should be in the flow-through).
Once you've bound, washed, and eluted your protein and confirmed it's purified to a relatively high level, THEN you can go about concentrating and dialysing it.
Echoing Hector: the second gel does not look like a purified, partially degraded protein prep. It looks like cell lysate: there seem to be a whole ton of different proteins, several of which are higher molecular weight than your protein of interest, which rather rules out degradation products.
And to be fair, protein degradation does not usually produce a neat 'ladder' of fragments of decreasing size (even in the presence of several different proteases), as most common proteases cleave at discrete, sequence dependent sites. A 66 kDa protein might degrade into two large fragments of 30 and 36, which themselves might eventually degrade into 10, 20, 10 and 16, and so on. The degradation patterns of purified proteins are often fairly distinctive.
Thanks Ditte Lundvig for your reply..
I will explain my workflow: Lysis of cell pellet - loading into HIS tag column for purification - elution into different fraction - mix all fraction and dialysis agaisnt required buffer - concentrating the sample
If your protein is 39KDa and 66Kda is what the reducing gel is showing then I guess there are other contaminants . If the protein degrades there are generally multiple bands corresponding to the same proteins of lesser size than the protein. As in you should be seeing a ladder like band pattern less than 39 Kda. is your protein a protease itself ? if so there could be some self cleavage issues .
No anirudh my protein is not a protease and predicted mol wt of my protein is 39kDa but in sds it shifts to 66kDa
What percentage gel are you using ? and do you heat your sample before loading ? what is your dye composition ? Do check with your ladder . I find it strange that something that has degraded gives higher size on the gel.
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