Gel Shift (EMSA) Protocol

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℃.