Strange "V" band in Gel Shift - (Aug/29/2007 )
I am using EMSA kit to do gel shift.
After I add protein to the DNA, the band looks like a "V". Any one has seen that? How can I get a pretty band?
PS: I run gel for 2h at 80V at RT.
This is the picture.
How old are the reagents? Acrylamide stocks? Buffer?
What buffer are you using? Try changing it.
The bands look "smeary".
Is there dI-dC in the rxn? Is it enough?
Using detergents often help to get sharper bands.
I agree with the last post that fresh reagents are best.
Sometimes higher NaCL conc. has positive effect (long shot).
If you are using loading buffer, do not use any. There's enough
glycerol in the rxn to load it.
Also, titrate each reagent to achieve optimal conditions.
I feel that you should try to make a drastic reduction in the amount of glycerol in your binding buffer. over-application of glycerol to gel shift samples will cause this smiley effect - and it's in both your binding buffer and your blue juice, don't forget