Gel Shift (EMSA) Protocol

There 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

³²P-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 ml TEMED

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 ml):

5X EMSA buffer: 4

Water: to a final volume of 20 ml

PolydI/dC: 1

BSA: 1

32P probe: 1 (10K cpm)

protein: 1-3 ml

let the reaction stand for 10-15 min at room temp., then load 18 ml per 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 C.