Hi,
I´m doing recombinant expression of a scFv with a his-tag in E.coli BL21-AI with T7-promoter. The expression is induced by arabinose. I separated inclusion bodies and supernatant after sonication, and purified both by Ni-NTA affinity chromatography. I also did SDS-PAGE on both supernatant and inclusion bodies. I need some help to interpret my results.
Here are my questions:
1. The bands of expected size are stronger on the gel from the supernatant than the one from the inclusion bodies. I expected it to be the other way around. Does anybody have any idea of why?
2. Bands from supernatant are also decided (weak bands above and over expected size). What can this indicate?
3. The bands from the inclusion bodies are a bit higher on the gel (about 10kDa bigger than the ones on the supernatant-gel). Why?
4. I used UREA to elute rests of the protein on the Ni-NTA column. The SDS-PAGE from proteins with UREA-treatment are formed as a big lump (but at the expected size of the scFv). What is this big lump?
5. There are smear in the wells of the bands which looks OK compared to expected size. What can this indicate?
Upper picture = gel from Inclusion bodies
Lower picture = gel from supernatant
In advance, thank you.
Best,
Sigrid.
What is your vector?
It seems to me, you have a vector expressed recombinant pro in secretive form thus there is a signal peptides can be explanation for your result.
About additional band, they maybe i.e dimer form of your pro or a pro of bacteria has affinity for Ni.
And I didn't understand your point of lump.
Regards
Thank you for the answer!
The lump: In the upper picture, in lane 52 and 53 there is a very conserved area of blue staining just obove 25 kDa. These proteins are eluted with UREA-buffer.
I just wonder why the bands are so conserved.
Best,
Sigrid
That's bands have the same MW with your target?
If the answer is yes, most likely those are your pro and so you have to play with con of imidazole.
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