Analysis of DNA-Binding Activity in Neuronal Tissue with the Electrophoretic Mobility-Shift Assay - Drugs of Abuse: Neurological Reviews and Protocols - page 310

2. Materials
2.1. Annealing of Oligonucleotides

1. Annealing buffer: 10 m M Tris-HCl, pH 7.5-8.0, 50 m M NaCl, and 1 m M
EDTA.

2.2. Radiolabeling of the Enhancer
1. 100 m M Dithiothreitol (DTT) is prepared in distilled water and stored at -20°C.
2. NucTrap Push columns are available from Stratagene, and can be substituted
with any other type of prepacked size-exclusion chromatography column that
retains nucleotides and short oligonucleotides to separate them from larger
pieces of DNA or RNA.
3. 1X STE buffer: 100 m M sodium chloride; 20 m M Tris-HCl, pH 7.6; and 10 m M
EDTA in distilled water.
2.3. Preparation of Protein Extracts

1. Sonication buffer for quick and easy protocol (100 mL): 2 mL of 1 M N -(2-
hydroxyethyl) piperazine- N -(2-ethanesulfonic acid) (HEPES)-KOH buffer, pH 7.9

(20 m M ), 25 mL of 100% glycerol (25%), 25 mL of 2 M KCl (0.5 M ), 150 µ L

L of 0.5 M sodium EDTA, pH 8.0 (0.2 m M ).
Bring to 100 mL with distilled water. Additives: Before use add to an aliquot of
sonication buffer (e.g., 1 mL): 1

of 1 M MgCl 2 (1.5 m M ), and 40 µ

L of 1 M DTT/mL (1 m M ) and 1X mammalian
protease inhibitor cocktail (commercially available from Calbiochem; cat. no.
539134) ( see Note 1 ). If state of phosphorylation is important, add 1

µ

µ L of

1 M NaF/mL (1 m M ), 1 µ L of 5 µ M okadaic acid/mL (5 n M ), and 1 µ L of

100 m M Na 3 VO 4 /mL (100 µ M ).

2. Buffer A for crude nuclear extract (100 mL): 1 mL of 1 M HEPES-KOH, pH 7.9
(10 m M ), 150 L

of 0.5 M sodium EDTA, pH 8.0 (1 mM), and 25 mL of 100% glycerol (25%).
Bring to 100 mL with distilled water. Additives: Before use add to an aliquot: 1

µ L of 1 M MgCl 2 (1.5 m M ), 500 µ L of 2 M KCl (10 m M ), 200 µ

L
of 1 M DTT/mL (1 m M ) and the 1X mammalian protease inhibitor cocktail
( see Note 2 ).

µ

2.4. Preparation of the Gel

1. 10X TBE buffer (Tris-borate-EDTA): 108 g of 0.89 M Tris base, 55 g of 0.89 M
boric acid, and 7.4 g of 20 m M EDTA disodium salt in 1 L of distilled water;
adjust pH to 8.3.

2. 10% Ammonium persulfate (APS) in distilled water: 1 g of APS in 10 mL of
distilled water. Lasts approx 1 mo at 4°C.

3. 30% Acrylamide- bis -acrylamide solution in distilled water (37.5 1): 29.2 g of

acrylamide and 0.8 g of bis -acrylamide in 100 mL of distilled water.

2.5. EMSA

1. 5X EMSA buffer: 50 m M HEPES-KOH, pH 7.9, 50% glycerol, and 0.5 m M
EDTA (from 0.5 M sodium EDTA, pH 8.0) ( see Note 3 ).