Mobility Shift Assay

Tue Dec 15 09:13:15 EST 1992

I've been trying to perfect the conditions for running a Mobility Shift Assay.
I'm using this assay to measure the DNA-binding activities of various proteins.
The probe is a 20 mer ds piece of DNA with 4 bases extension at each end and it
 is labled by kinasing. I have been runing the reactions in 4% acrylamide gels
in 0.5xTBE, at 50-100 V at 4 C (cold room). The bands I'm getting are smeared
and smilling (though I'm not).
Could I please have your comments regarding the following points:
1. Presence of protruding ends on the labled oligo
2. Kinasing vers. filling-in for labaling
3. Poly d(I-C) as non-specific competitor vers. sonicated calf-thymus DNA
4. The use of 0.5 x TBE as running buffer, and running conditions in general
5. Your chosen loading buffer
6. Any other tricks?
Thanks in advance!!!

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