I made recombinant fusion protein by DNA cloning.
C-term of my target protein has some tag for detection and purification.
For example, Targert- His6-GST
When I did western blot analysis using Anti-GST. It shows on the film.
But When I use His6 antibody, I cannot see correct size of band.
To whether antibody problem or not, I did experiment for positive control and negative control.
As conclusion, Antibody has no problem.
In this case I cannot under stand why I cannot see any band when I use His6 antibody.
When we did SDS-PAGE, as another experiment, samples were boiled and add DTT.
Can you explain why this kinds of problem occur?
Hello, Seo
Did you confirm by sequencing that the His tag is in fact in your construct? Another suggestion is to put the His tag to the N-terminus, i.e. make His-target-GST construct. I would think about the problem of folding and/or accessibility of the His epitope between your target and GST.
Good luck!
Hi Seo,
Which kind of anti-His Ab did you use? There are commercialized Ab with anti-N-His or anti-C-His activity, you may need to check the datasheet of anti-His Ab. In addition, I'm not sure whether these anti-His Ab can recognize internal His tag like your case.
Good luck
Hello Seo,
The absence of the signal in WB can be also caused by the inaccesibility of the epitope to Abs. During the detection steps, the protein, which was denatured in SDS-PAGE, can (at least partly) renature on a membrane and the epitobe (especially internal but also the external one) can be buried in the renatured (part of) protein.
Ela
It is possible that you have lost the Hist tag or you have not loaded sufficient protein per lane to detect.
You could try purifying/binding to IMAC column from cell lysate.
All the answers above give a bunch of possibilities of what might have happened. Another option is the sensitivity of the anti-His antibody that you are using - you might need to use higher antibody concentration to get approximately the same signal intensity as with anti-GST.
Other answer I already tried all of them.
IMAC, antibody sensitivity test, sequencing, and so on.
But I didnt consider the possibility of renature of protein.
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