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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 Title | Procedure | Solutions | BioChemicals | Hints | Printable Version
Contributor:	The Laboratory of Steve Hahn at the Fred Hutchinson Cancer Research Center
Procedure Title | Procedure | Solutions | BioChemicals | Hints | Printable Version
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).
Solutions Title | Procedure | Solutions | BioChemicals | Hints | Printable Version
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 Title | Procedure | Solutions | BioChemicals | Hints | Printable Version
Poly [dG-dC] Bovine Serum Albumin Bisacrylamide Dry Ice TEMED Glycerol Bromophenol Blue Ammonium Persulfate Tris Potassium Chloride Acrylamide Magnesium Chloride Acetic Acid p[dG-dC] Glycine DTT
Protocol Hints Title | Procedure | Solutions | BioChemicals | Hints | Printable Version
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.