the electrophoretic mobility shift assay (emsa) - Microbiology

17 downloads 11 Views 60KB Size Report

THE ELECTROPHORETIC MOBILITY SHIFT ASSAY (EMSA) (OR GEL SHIFT TO ITS CLOSE PERSONAL FRIENDS) Radiolabeled DNA Probes Annealing Oligonucleotides Complimentary oligonucleotides which contain DNA binding element sequences are typically synthesized with a 5’-XhoI/SalI (5’-tcga-3’) overhang which facilitates both radiolabeling with Klenow and cloning into reporter vectors. Any 5’ overhang with a G in the overhang—i.e. not EcoRI—will work. Dissolve the oligonucleotides in DDI H2O to a final concentration of 100 µM (preferred) or 1 mg/ml prior to use. The final concentration of annealed oligonucleotides should be 0.5 mg/ml or 25 µM. To anneal oligonucleotides make a reaction that contains: + Strand Oligonucleotide - Strand Oligonucleotide 10x Oligo Annealing Buffer DDI H2O Total

25 µl 25 µl 10 µl 40 µl 100 µl

Heat the above reaction as follows: 95°C 2 min A°C* 5 min A°C→37°C 90 min 37°C 2 min 4°C Hold * A°C is 5°C over the Tm of the oligonucleotide (at 50-100 mM salt) Oligonucleotide Labeling Mix (in order)… Component [Stock] EcoPol Buffer (NEB) 10x dNTP Labeling Cocktail* 10 mM DDI H2O 32P-a-dGTP (3000 Ci/mMol, 1mCi/ml) Annealed Oligonucleotides 0.5 µg or 25 µM Klenow DNA polymerase 5U/µl Total *

Vol. 10 µl 1µl 76 µl 5 µl 4 µl 2 µl 100 µl

10 mM of each dATP, dCTP, and dTTP if labeling with dGTP (omit whichever dNTP you are labeling with).

November 13, 2003 Edition

Page 1

1. Incubate reaction 20 min. at room temperature in a shielded acrylic rack. 2. Meanwhile, fill a spin column with TE washed Sephacryl S-200HR. Let settle ~ 1cm and refill. Place in a 50 ml Falcon tube (can be reused if not contaminated with radioactivity). Centrifuge in IEC tabletop clinical centrifuge at speed 6 for 2 min. Discard liquid from reservoir. 3. Apply labeling reaction to center of spin column dropwise. Spin in IEC tabletop clinical centrifuge at speed 7 for 5 min. 4. Aliquot labeled oligonucleotides into µfuge tubes and store at –20°C in a shielded box. Count probe by adding 5 µl of labeled probe to a 0.5 ml µfuge tube. Cap the tube and cut off the hinge. Place the tube in a scintillation vial. Count in a scintillation counter. The scintillation vial can be reused.

The Actual Gel Shift Assay The Gel Pour a 5% acrylamide (29:1)/0.5x TBE Gel. 40% Acrylamide (29:1 Ac:Bis) 10xTBE DDI H2O 30% APS TEMED

5 ml 2 ml 33 ml 100 µl 35 µl

(EM Science#1700)

Make ammonium persulfate (APS) at least every month or so and store at 4°C. Add APS and TEMED immediately before pouring the gel to initiate polymerization. Receptor Dilution Dilute nuclear receptors (usually either purified GST proteins or baculovirus produced insect cell nuclear extracts). This usually done by doing a titration of a receptor preparation to determine the amount of receptor required to bind approximately one-fourth to one-third of the probe. Dilute the probe in NED Buffer. Dilute the receptors immediately before use and keep on ice until use.

November 13, 2003 Edition

Page 2

The Binding Reaction 1. Make a reaction consisting of the following… 1x Binding Buffer 2.5 mg/ml BSA 20 ng.µl poly dI•dC 1 mM DTT Probe* Nuclear Extract Dilution† DDI H2O

5x 50 mg/ml 2 mg/ml 1M


2. 3. 3. 4. 5. 6. 7.

4 µl 1 µl 0.2 µl 0.02 µl 4 µl 2 µl to 20 µl

Typically a final probe concentration of 2 pmol (100nM final) and 250,000 counts in 4 µl are used. † Typically extracts are diluted so that 2 µl with shift 1/4-1/3 of the probe. Do not use more than 5µl as excess salt will begin to interfere with binding. Incubate approximately 15 min. at room temperature. Prerun gel at 180V for at least 10 min. Add 3 µl of 10x Native sample dye to the free probe reaction and 3 µl of 25% Ficoll 400 to each other reaction. Centrifuge briefly in a radioactive centrifuge. Load 20 µl of each reaction/well. Run the gel at 180V for approximately 90 min—this will prevent the unbound probe from running off the gel and making the bottom buffer chamber radioactive. It may be run longer if needed. Dry the gel on Whatman 3MM filter paper at 80°C for 1 hr on the vacuum gel dryer. Expose overnight on a Phosphorimage screen and analyze as needed.

November 13, 2003 Edition

Page 3

Solutions: 10x Oligo Annealing Buffer Component [Stock] 100 mM Tris HCl (pH 7.5) 1 M 1 M NaCl 5M 10 mM EDTA 0.5 M DDI H2O

Vol. 1 ml 2 ml 200 µl 6.8 ml

5x Binding Buffer 50 mM Tris•Cl, pH 7.5 10 mM MgCl2 250 mM KCl DDI H2O

1M 1M 2.5 M

5 ml 1 ml 10 ml 84 ml

2x NED Buffer 20 mM HEPES, pH7.9 10% Glycerol 300 mM KCl DDI H2O

NED Buffer (1x)

1M 50% 2.5 M

80µl 400 µl 480µl 1040 µl

2x NED 50 mg/ml BSA 1 M DTT DDI H2O

250 µl 100 µl 0.5 µl 150 µl

10x TBE Electrophoresis Buffer

Tris (free base) Boric Acid 0.5 M EDTA, pH 8.0

1 liter

2 liters

4 liters

6 liters

108 g 55g 40 ml

216 g 110 g 80 ml

432 g 220 g 160 ml

648 g 330 g 240 ml

10x Native Sample Dye Bromophenol Blue Xylene Cyanole Glycerol DDI H2O

November 13, 2003 Edition

Page 4

0.02 g 0.02 g 5 ml 5 ml

Suggest Documents