I have been doing EMSA using Pierce kit for 4 months. My band shifted without adding poly dI-dC.
But with poly dI-dC, I didn't see no shift at all. I add 50ng/ul final concentration for poly dI-dC. I used 4ug of nuclear extract.
Can I leave this non-specific competitor in my reaction and go on to supershift?
I labeled my oligo with Pierce 3' end biotin labeling kit. I got the nice free probe every time.
My protocol is
- Incubate reaction at RT for 20 min (buffer, MgCl, glycerol, poly dI-dC in each reaction)
- 6% non-denaturing gel
- Run and transfer to Millipore nylon membrane with 0.5%TBE, (pre-chilled)
- detect biotin labeled oligo with non-radioactive Pierce kits.
Thanks in advance for replying.
Edited by N_L, 31 August 2009 - 06:35 PM.