Mobility Shift Assay

[Jorge L. Sepulveda]

In article <1992Dec15.145242.3047 at gserv1.dl.ac.uk> 
LCNAVOT at WEIZMANN.WEIZMANN.AC.IL (Nir Navot) writes:
> 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!!!
> Nir

Nir,

Here are a few tricks,

1. I always purify my ds oligos, either after annealing or after 
filling-in with Klenow; if you choose the hot nucleotide as the last to be 
filled in, you know that all you labeled probe is double stranded and 
blunt ended (although the incorporations are lower, but I use only 10,000 
to 20,000 cpm/lane)
2. Maybe for the above reason, I find filled in probes to have much less 
background than end-labeled
3. For some probes, 1 5g/reaction of E. coli DNA is better, for others I 
use 1 5g/5l of dIdC
4. I use 1/4 TBE and run it at 10W, 4!C
5. I just add dyes (XC+BB) to the sample immediatly before loading, 
because there is enough glycerol to sink the sample
6. Be carefull with the order of addition of reagents; I do buffers, 
competitor dna, nuclear extracts, wait 5-10', add hot probe
7. The amount of glycerol (may be because it interacts with borate) can 
cause a lot of smearing and background; keep it less than 4% in the 
reaction

Cheers, 




Jorge L. Sepulveda, 

Dept. of Pathology,                        Email: Jorges at bcm.tmc.edu
Baylor College of Medicine                 Telephone: 713-798-7330
One Baylor Plaza, Houston, TX 77030        Telefax: 713-798-5838