EMSA Protocol

Pour Acrylamide Gel:

1. Assemble Plates, spacers, and clamps. Seal with 1% agarose to prevent leaks.

2. Pour 5% acrylamide Gel

Plate size Large Medium

H2 O 78mL 39mL

10x TBE 5mL 2.5mL

30% Polyacrylamide 16.6mL 8.4mL

10% APS 500uL 250uL

mix well while minimizing bubble formation. Add 50uL/25uL TEMED. Mix and pour, add combs. Gel will take 30-45 min to polymerize. After polymerization, gel can be wrapped in Saran Wrap and stored at 4o C.

5x Binding Buffer:

Composition Recipe for 10mL

50mM Tris HCl (pH 8.0) 0.5 mL of 1M Tris HCl (pH 8.0)

750 mM KCl 3mL of 2.5 M KCl

2.5 mM EDTA 50uL of 0.5 M EDTA (pH 8.0)

0.5% Triton-X 100 50uL Triton-X 100

62.5 % glycerol (v/v) 7.87 g glycerol

1mM DTT add DTT fresh before use

Binding Reaction:

1uL of poly-dIdC (1ug/uL in TE)

2uL of 5x Binding Buffer

1uL of labeled probe

1uL cold competitor (if needed)

0.1uL 100x BSA

?? uL Nuclear Extract (5ug protein total)

Add H2 O to 10uL

Incubate 30 min room temp. Add antibody for supershift (if needed). Incubate additional 30 min, room temp.

While binding reaction is incubating, pre-run polyacrylamide gel 150V, 30 min, using 0.5xTBE as running buffer.

Run samples on acrylamide gel ~2hours 150V

Dry the Gel (Optional)Dry the Gel (Optional)

Transfer Gel to Whatman Paper. Cover top of gel with Saran Wrap and dry at 80o C in vacuum dryer 1-2 hour.

Expose the Gel

Place gel in cassette with reflection screen. Add film and place in -80o C freezer.