Electrophoretic Mobility Shift Assay (EMSA)
Preparation of nuclear extracts
[All steps must be performed on ice.]
1. Harvest cells (on 100-mm culture dish) using 1 mL of PBS to microcentrifuge tubes
[2,500 rpm, 4 min, 4 C].
2. Wash the cells with PBS and combine two 100-mm culture dishes per sample
[2,500 rpm, 4 min, 4 C].
3. Resuspend the cell pellets with 1 mL of Lysis buffer and stand for 5 min on ice
* Lysis Buffer: 10 mM Tris-HCl(pH 8.0), 60 mM KCl, 1 mM EDTA, 1 mM
dithiothreitol, 100 M PMSF, 0.1% NP-40
[Homogenize ~100 mg tissues with 1 mL of Lysis Buffer and stand for 5 min on ice]
4. Centrifugation [2,500 rpm, 4 min, 4 C]
5. Wash the nuclear pellets with 1 mL of Lysis buffer (without NP-40)
[2,500 rpm, 4 min, 4 C]
6. Resuspend the nuclear pellets with 50 ~ 100 L of Nuclear Extract Buffer, stand for 10
min on ice, vortex briefly, and centrifuge [14,000 rpm, 15 min, 4 C]
* Nuclear Extract Buffer: 20 mM Tris-HCl(pH 8.0), 420 mM NaCl, 1.5 mM MgCl2,
0.2 mM EDTA, 25% glycerol
7. Obtain supernatant (“Nuclear extracts”), aliquotes (10 ~ 20 L), freeze quickly in dry
ice or liquid nitrogen, and store in deepfreezer (-80 C) before use
[Determine protein concentration of nuclear extracts (4 L) by Bradford assay]
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Dr. Lee’s Lab
Gel Shift Assay
1. Preparation of 5% polyacrylamide gel:
DW
7.5 mL
2X Electrophoresis buffer
10 mL (0.5X TBE)
40% acrylamide/bis solution
2.5 mL
10% ammonium persulfate (APS)
200 L
TEMED
20 L
Solidify for at least 1 h and pre-run for 2 h at 150 V.
2. Preparation of gel loading sample:
14 L of master mix [DW, 8 L + 5X Binding buffer, 4 L + poly(dI-dC), 2 L (2
g)] + Nuclear extracts 4 L (2 ~ 6 g protein)
* 5X Binding buffer: 50 mM Tris-HCl(pH 7.5), 5 mM EDTA, 0.5 mM DTT,
50% glycerol
3. Incubate for 10 min at room temperature (RT)
4. Add 1 L of competitor (unlabeled oligonucleotide) to “Inducer group” and further
incubate for 15 min at RT
5. Add 2 L (40,000 cpm) of 32P-labeled oligonucleotide probe and incubate for 25 min
at RT
6. Load 10 L of sample to 5% polyacrylamide gel and run for 3 h at 150V
[Gel running buffer: 0.25X TBE (pH 8.0)]
7. Dry gel & autoradiography
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Dr. Lee’s Lab
Preparation of Buffer:
Lysis Buffer (40 ml preparation) [with or without NP-40]
Stock solution
Final Concentration
Volume
1 M Tris-HCl
10 mM
400 l
1 M KCl
60 mM
2.4 ml
500 mM EDTA
1 mM
80 l
1 M DTT
1 mM
40 l
100 M
40 l
0.1%
400 l]
100 mM PMSF
[10% NP-40
D.W.
Adjust to 40 ml
Nuclear Extract Buffer (10 ml Preparation)
Stock solution
Final Concentration
Volume
20 mM
200 l
5 M NaCl
420 mM
840 l
1 M MgCl2
1.5 mM
15 l
500 mM EDTA
0.2 mM
4 l
25%
5 ml
1 M Tris-HCl
50% Glycerol
D.W.
Adjust to 10 ml
5X Binding Buffer (10 ml Preparation)
Stock solution
Final Concentration
Volume
1 M Tris-HCl
50 mM
500 l
1 M DTT
0.5 mM
5 l
500 mM EDTA
5 mM
100 l
100% Glycerol
50%
D.W.
5 ml
Adjust to 10 ml
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Dr. Lee’s Lab