Gel shift - (Sep/22/2006 )
I am doing gel shift assay now. The protein I use is about 16KD and probe (oligo) is 19bp. The interaction between them seems to be very strong because shift can be observed clearly by EB staining (20ul binding system with 4ug protein and 4ug probe). But when I try this assay with radioactive labeled probe (20ul binding system with 100ng protein and 1ng probe), free probe can be seen but no shift can be seen. The complex is stuck in the well ! What's wrong with my assay? I use 10% PAGE and do not use poly(dI-dC) in the binding system. Dose this affect the result?
Thanks for your attention!
-biomaomao-
QUOTE (biomaomao @ Sep 22 2006, 06:24 PM)
I am doing gel shift assay now. The protein I use is about 16KD and probe (oligo) is 19bp. The interaction between them seems to be very strong because shift can be observed clearly by EB staining (20ul binding system with 4ug protein and 4ug probe). But when I try this assay with radioactive labeled probe (20ul binding system with 100ng protein and 1ng probe), free probe can be seen but no shift can be seen. The complex is stuck in the well ! What's wrong with my assay? I use 10% PAGE and do not use poly(dI-dC) in the binding system. Dose this affect the result?
Thanks for your attention!
Thanks for your attention!
could it be that your probe is not 100% radioactive labeled, that's why you see a shift with EB (you see 100% of the shift), but nothing by autoradiography, because you can only see a small fraction of the radioactive labeled probe, (not enoughÃ
?
-Missele-