I am using Novagen kit for r-protein purification and I have a smear in my SDS -PAGE after purification, do I need a post purification treating of my r-protein for long storage ??
I have no prior experience on R protein, nor knowing which kit you used, nor knowing the protein expression system. Thus my comments would rather general, apology. I hope it may help, though.
I think you should first check for the nature of your protein. is the smear characteristic to your protein (perhaps from glycosylation)? Depending on which expression system employed, you might get different glycosylation profile of your gene product (they may migrate slightly different on PAGE). Bacterial system (i.e. E. coli) cannot perform post translational modification but you still have a possibility of having native-like protein (which might migrate slightly different on PAGE, if the reducing/denaturing agent was not strong enough to completely unfold). Perhaps the smear indicating presence of contaminant(s) with size(s) close to your R-protein? Further, does your R-protein stand for freezing and thawing?
One of my students purified an enzyme that demonstrated potential thermal stability. Upon thawing after freezing, the protein band has smear on a PAGE analysis. Sample stored at 4 C on the other hands remained nicely one thick clear band. From that finding, we store the protein at 4 C (not frozen) instead.
What is the stability of your favorite protein? Did you run it promptly after purification? meanwhile the entity of your protein is important as well. If everything goes well you must be check the protocol of SDS-PAGE procedure. Bests
Wangsa Tirta Ismaya, Thank you so much for your help, My protein is a part of IgT in fish, I use the mammalian expression system hek 293 , I use iodoaetamide for irreversible reduction but the smear appears both with and without the iodoacetamide after incubation with SDS for 10 minutes at 85 C, I did not check the stability after freezing and thawing , it worth to try DSD-PAGE directly after purification. Thanks a lot for help.
Eskandar Omidinia, what you mean by stability ? you mean heat stability, I do not know this is the first time to use heck 293 for production of a part of IgT in salmon, I do not understand what promptly means but actually we use the direct blotting.
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