I kept getting double bands when western blotting, one at 25kDa and one at round 30kDa. I thought it could happen because I know that there are two isoforms from this gene. problem cames in here that when si-transfection is performed only one of the two bands is disappeared. Does that mean siRNA affected only one of two isoforms? or possiblely high non-speific binding?
hi
you can check it (if you have gene sequence ) with WB :
1-test again with positive and negative cell lysate( Your desired antigen) , then if you see both bands in the negative sample, this is for the test background and not the new transcript.
2- check the si-RNA sequence design for N-term or C-term of your protein ?
good luck
The sequence of the two isoforms should be known.
If your siRNA is directed against both isoforms, it is unlikely that only one transcript is affected.
In case only one transcript is targeted, you have to design a second siRNA.
More likely, your antibody/serum cross-reacts with another protein. I would recommend to try an independent generated antibody. It is unlikely that this antibody recognizes the same "unspecific" additional band.
have you checked the membrane without primary antibody (control for the secondary)?
- Badges
- Science method