Electrophoresis Mobility Shift Assay (EMSA)
1. Make double-stranded oligos
10µM ds-oligo add
10µl of 100µM oligo A
10µl of 100µM oligo B
80µl of STE Buffer
Heat at 94-95°C for 1 min in heat block then unplug heat block and let oligos reach room
temp ~ 60 min (STE Buffer 10mM Tris pH8.0, 50mM NaCl, 1mM EDTA)
2. Label ds-oligos with T4 polynucleotide Kinase (Gibco BRL) in forward rxn.
1µl 10µm ds-oligo
5µl γP32 ATP
4µl 5X T4 Kinase Buffer
1µl Kinase
9µl H2O to 20µl
20 µl total
Incubate at 37°C for 30 min, inactivate Kinase by heating for 10 min at 65°C
Purify probe by non-denaturing acrylamide gel
Non-denaturing Gel (Small gel cast)
4 ml acrylamide (19:1)
1 ml 10X TBE
5 ml H2O
112 µl 10% APS
5 µl TEMED
10 mls
Run in 0.5X TBE (25mls TBE/ 475mls H2O) 40 min 100 V
After gel is run wrap in plastic wrap. Expose gel to film (5 to 10 min) to locate
and identify labeled oligo. Mark gel and cut out band. Remove plastic and crush gel in
1.5 ml microcentrifuge tube and add 0.6 ml of 0.3M NaAc and let rock 3hrs to overnight.
Spin for 1 min in microcentrifuge and pipette off 0.5 ml of supernatant solution, taking
care to avoid polyacrylamide pieces. Add 2.5X EtOH (1250µl) and place in -80 for at
least 10 min. Spin at 4°C for 10 min. Remove EtOH/NaAc solution and keep pellet.
Vacuum dry and resuspend in 50 µl of H2O.
3. Binding Reaction
Duc
yan
10 mM HEPES pH7.9 13 mM HEPES
1 mM EDTA
0.7 mM EDTA
1 mM DTT
0.3 mM DTT
1 mM KCl2
40 mM KCl2
1 mM MgCl2
10% Glycerol
7% Glycerol
1µg polydI-C
1 µg polydI-C
0.3 µg/µl BSA
5 µg BSA
GST-fusion protein
protein
H2O
water
______Probe
Probe_____
25 µl total
18 µl total
pntp2
4.4 mM Tris pH7.5
0.7 mM EDTA
0.3 mM DTT
40 mM KCl2
9% Glycerol
1 µg polydI-C
5µg BSA
protein
water
Probe______
18 µl total
Incubate at RT for 30 min (37°C,15min). Add 1µl loading dye and load onto 5%
non-denaturing acrylamide gel (large cast). Run @ 200V, 30mA for 10 wHours
(about 2 hrs) in 0.5X TBE (50mls TBE/ 950mls H2O)
Dry gel on Whatman paper
Expose gel (depends on how hot)
Non-denaturing Acrylamide Gel for Gel Shift (60 mls)
8 ml acrylamide (40:1)
3 ml 10X TBE
3 ml Glycerol 50%
450 µl 10% Ammonium persulfate
70 µl TEMED
45.5 H2O
60 mls