EMSA Protocol
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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.