Dear all,
I am expressing a virus protein about 9 kDa on a 16% tricine gel (150V constant). I induced protein expression with 0.5 mM IPTG and collected the cells pre- and post- induction.
The size of the expected band in the 3rd host seems to be similar in size to the same protein when expressed at other conditions. However, I cannot conclude which band is my protein of interest (Lane 1, 2, or 3). The question is, are there any explanations why my protein is expressed differently?
Thanks in advance for all the answers.
P.S.1 All the proteins are from the same gene and construct.
P.S.2 Sorry about the markers, they were a bit diffused. I added the markers in red which I compared the sizes with my other gels.
Dear Pongpawan,
Firstly, you can check for your protein using a western blot. If you do not have antibodies for your protein I would suggest fusing it with a tag such as His. Since anti-His abntibodies are available commercially, you would be able to detect your protein's expression when induced.
It is only after that can you conclude that your protein is being expressed differently in the different strains.
Abhishek
ya first be sure if its being expressed in all the strains/hosts you are trying to screen. Blot is always a better answer for the crude lysates.
If it turns out that the identified bands from the different strains are all your protein, then it may be that it suffers a proteolytic cleavage in some strains.
I tried western blot and it was the same protein.
However, when I purified with Ni-NTA, it turned out to be all the same size and now I'm so confused which one to choose for large scale expression.
Pick whichever one expresses the most intact protein in a soluble form.
yes. check the intact tags on both the sides. check that which one gives you the best yield also.
good luck!
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