I expressed my recombinant protein in E.coli, BL21 (DE3) with a MBP fusion protein. But after cut by protease to remove the MBP part, I found my target protein size is less than I expected, but the protease protein from the reaction show correct size after run SDS page instead of my target protein. So should I believe the SDS gel?
Hi
I didn't do much SDS-PAGE, but in my experience, don't mind it if the band is a bit lower than the marker.
And check your marker, sometimes the bands of the same marker are a little different in the different SDS-PAGE, such as SeeBlue2 in tricine or glycine.
Dear Wang Yixia,
Thanks for your idea. I agree with you about the marker drifting a little bit in different gel, since it happened in my experience. But for my trouble, the size of my target band seems like drift around 10kDa, that's too much I guess. So I'm thinking about Jiang Kunpeng's idea about the degradation. I checked my protein sequence and there is no cleavage size for the protease that I used. If it really happened from other outside contaminated protease, maybe I should better try to modified my protocol for purification. So far, I just want to get more idea about the possibility from SDS-Page gel. Thanks both of your information.
Hi,
Unfortunately sometimes its happens with MBP-fusion proteins. MBP is a huge protein and applicates for production small proteins very well. If target protein MW is more than 20 - 30 kDa mRNA translation could be unfull. So you can see a band which is seems like a smaller protein.
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