Mirmira Laboratory at the University of VirginiaThere are multiple variations to this protocol, but we find that this one works well in all cases we tested.
Reagents:
5X EMSA Buffer:
50mM HEPES (pH 7.9)
375 mM KCl
12.5 mM MgCl2
0.5 mM EDTA
5 mM DTT
15% Ficoll
32 P-labeled oligonucleotide probe
polydI/dC: 1 mg/ml in TE
BSA: 10 mg/ml in TE
5% Polyacrylamide gel (30 ml)
22 ml water
3 ml 5X TBE
5 ml 30% Acrylamide
0.210 ml 10% Ammonium persulfate
10-100 mlTEMED
5X TBE (1 L):
54 g Tris base
27.5 g boric acid
20 ml 0.5 M EDTA (pH 8.0)
Reaction:
Combine the components in the following order (in m l):
5X EMSA buffer: 4
Water: to a final volume of 20 m l
PolydI/dC: 1
BSA: 1
32P probe: 1 (10K cpm)
protein: 1-3 m l
let the reaction stand for 10-15 min at room temp., then load 18 mlper lane on a 5% polyacrylamide gel. Run at 150 V for 2h at room temperature, then dry the gel and expose 4-16 h to film at –80℃.
