Gel Shift with Glutaraldehyde

Sunday, October 19, 2003

Description
Gel Shift with Glutaraldehyde

Procedure
1. Pour a 4% acrylamide high ionic strength Tris/Glycine gel that is 1.5 mm thick with a 12 to 14 tooth comb. Combine: 6.7 ml 30 % Acrylamide (60:1), 10 ml 5X Tris/Glycine Buffer, 33.3 ddH2O, 250 l 10% Ammonium Persulfate, and then 60 l TEMED.

2. Set up and electrophoresis the gel for at least 1 hr at 100V in 1X Tris/Glycine Buffer.

3. Make up the radioactive mix on ice with about 2 fmol labeled DNA per reaction: 0.4 l 100 mM GTP/reaction, 0.4 l 100 mM ATP/reaction, 2 l 5X Binding Buffer with DTT/reaction, 0.1 l 20 mg/ml Bovine Serum Albumin/reaction, add ddH2O to final total reaction volume of 5 l.

4. Thaw the extract to be analyzed on ice. Set up a 10 l DNA-binding reaction on ice with the following additions in this order: ddH2O, Buffer (HGKED), 5 l Hot Mix, and 0 to 5 l protein extract.

5. Centrifuge the tube briefly at room temperature to mix (pulse in a microcentrifuge).

6. Incubate for 15 min at 20C to bind the DNA.

7. Add 1 l of 0.1% Glutaraldehyde to the side of the tube.

8. Pulse in a microcentrifuge and place tube on ice immediately.

9. Add 1 l Glutaraldehyde to quench the crosslinking reaction and pulse in a microcentrifuge to mix and place immediately on ice.

10. If you wish to run a supershift analysis, add about 40 ng of affinity-purified antibody in 1 to 2 l volume to each tube.

11. If running a supershift analysis, incubate at room temperature for 10 min to bind the antibody.

12. Rinse out the wells (i.e. "blow out" the wells) of the gel and load the sample and run the gel for 2.75 to 3.5 hr at 100V. Dry the gel for 30 min and expose it to X-ray film.

Recipes
HGKED Add DTT fresh before use
Add KCl to desired concentration
20 % Glycerol
0.5 mM DTT
0.5 mM EDTA
20 mM HEPES, pH 7.6 with KOH

20 mg/ml Bovine Serum Albumin

100 mM ATP

100 mM GTP

10 % (w/v) Ammonium Persulfate

30 % Acrylamide 60:1 acrylamide:bisacrylamide (This is not for protein gels)
Caution! see Hint #1

0.1 % Glutaraldehyde Freshly dilute an aliquot of commercial 25 % glutaraldehyde in ddH2O to 0.1 % (v/v) and keep on ice until use. (Caution! see Hint #1)

Glutaraldehyde quench 1 M Tris-HCl, pH 7.5
1 M Glycine

Binding buffer (5X) 10 mM EDTA
50 % Glycerol
add DTT to 2.5 mM just prior to use
250 mM KCl
100 mM Tris-HCl, pH 7.5

Tris/glycine buffer (5X) 2.7 mM EDTA (not the sodium salt)
380 mM Glycine
50 mM Tris base
pH 8.5

Supplies

Tips
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.

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Gel Shift with Glutaraldehyde