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MOLECULAR BIOLOGY: WORKING WITH PROTEINS
PROTEIN DETECTION: GEL ELECTROPHORESIS
GEL MOBILITY SHIFT ASSAY USING MAGNESIUM CHLORIDE AND EDTA
Gel Mobility Shift Assay Using Magnesium Chloride and EDTA
Contributor: The Laboratory of Steve Hahn at the Fred Hutchinson Cancer Research Center Procedure
A. Gel Shift Reaction
1. Prepare a 20 μl binding reaction by combining the following ingredients:
4 μl of 5X Binding Buffer
0.2 μl of 0.1 M DTT
2000-5000 cpm labeled DNA (see Protocol ID#1235)
0.125 μg of p[dG-dC]
ddH2O to a 20 μl final volume
Add proteins to the reaction last (see Hint #1).
2. Incubate the protein DNA solution at room temperature for approximately 30 to 40 min.
3. After incubation, load samples onto a Polyacrylamide Gel (see Section B below).
B. Observing a Gel Shift on the Polyacrylamide Gel
1. Prepare gel as follows:
10.5 ml of Acrylamide/Bisacrylamide solution
2.5 ml of 10X TGOE buffer
1.75 ml of 50% Glycerol
20.9 ml of ddH2O
0.3 ml of 10% Ammonium Persulfate
30 μl of TEMED
2. Cast the gel and set it in a cold room (4°C) approximately 5 to 10 min prior to loading the sample (refer to electrophoresis section of Protocol ID#520).
3. Wash the wells several times with 1 X TGOE (running buffer).
4. Pre-run the gel at 160 V for 15 minutes.
5. Wash the wells out several times with 1 X TGOE again.
6. Add the optimal volume of Loading Buffer to the samples (the ratio of sample to Loading Buffer should be determined empirically to ensure adequate dilution of the protein/DNA complex).
7. Begin the electrophoresis at 160 V.
8. Load the samples onto the wells of the gel. Start the gel immediately after loading the samples.
9. Electrophorese for approximately 45 min.
10. After completion of the electrophoresis, remove the gel and analyze the band shift by Autoradiography (see Protocol ID#9036).
Loading buffer 1X TGOE
0.05% Bromophenol Blue (see Hint #3)
35% (v/v) Glycerol
Gel Buffer 1X TGOE 50% (v/v) Glycerol Acrylamide/Bisacrylamide Solution CAUTION! See Hint #2
0.33% (w/v) Bisacrylamide
20% (w/v) Acrylamide
Prepare in ddH2O just before use
TGOE Buffer (10X) pH 8.2 using Acetic Acid.
0.25 M Tris
1.9 M Glycine
Binding Buffer (5X) OPTIONAL: add 25 μl of saturated Bromophenol Blue (approximately 0.1% in ddH2O per ml of 5X Binding Buffer (BioRad) (see Hint #3)
25 mM MgCl2
500 μg/ml BSA
300 mM KCl
Store buffer at -70°C.
20% (v/v) Glycerol
100 mM Tris-HCl pH 8.0
poly-[dG-dC]-Deoxyribonucleic Acid Protein Solution Proteins are typically stable during multiple repeated freeze-thaw cycles.
Thaw proteins quickly on ice.
(X) mg/ml protein (see Hint #1) in Protein Dilution Buffer.
Ammonium Persulfate 10% (w/v) Ammonium Persulfate
Prepare in ddH2O just before use
Protein Dilution Buffer 1 mM DTT
150 mM KCl
10% (v/v) Glycerol
50 μg/ml Bovine Serum Albumin
20 mM Tris pH 7.9
Store Dilution Buffer at -70°C.
0.5M DDT Prepare in ddH2O just before use 0.1M DDT Prepare in ddH2O just before use BioReagents and Chemicals
Bovine Serum Albumin
1. The amount of protein added must be determined empirically and will be based on the affinity of the protein to bind to the DNA
2. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
3. Bromophenol Blue may interfere with protein binding to DNA. The effects of the addition of Bromophenol Blue should be determined empirically.