EMSA
Electrophoretic Mobility Shift Assay (EMSA)
A. Annealing of oligos
1X TE Buffer: 10mM Tris, ph 7.5-8.0, 1mM EDTA
1.Dissolve the oligos to concentration 1ug/ul with 1x TE buffer
2.Mix complementary oligos at 0.5 ug/ul (each take 50ul) in TE-100mM NaCl
(add 1M NaCl 10ul) in 1.5ml tube, wrap the lid with parafilm , and lock cap with cap lock.
3.Put the tube into a 1L beaker containing about 300ml boiling water (heater is
okay), and boil for 10 min.
4.Put the tube in the beaker and cool down to room temperature. (about 1 hour)
5.Store the oligos in -20o C
B. Label the probe
1.Dilute the annealed oligos (500ng/ul) to 50ng/ul;
2.Denature 50 ng (1ul) of oligos dissolved in 6 ul of distilled water in a 1.5ml
microcentrifuge tube in 1000C 5 min in heater, then immediately cool on ice for 2min;
3. Perform the following additions on ice:
2 ul 0.5 mM dATP solution
2 ul 0.5 mM dGTP solution
2 ul 0.5 mM dTTP solution
15 ul Random Primers Buffer Mixture
6 ul (approximately 50 Ci) [-32P]dCTP, 3000 Ci/mmol, 10 uCi/ul
Add water and make total volume of 49 ul, then mix briefly;
4.Add 1 ul Klenow Fragment, mix gently but thoroughly, and centrifuge briefly.
(Or Add the Klenow Fragment first then add p32 dCTP);
5.Incubate at 25o C (room temperature is okay) for about 1.5 hours.
C. Purify the probe
1. Remove the column (Sephadex G-50 (fine) Quick Spin Columns (TE) for radiolabeled DNA purification) from the storage container, and gently invert it several times to resuspend the medium. (Shake it)
2. Remove the top cap from the column, then remove the bottom tip. This sequence is necessary to avoid creating a vacuum and uneven flow of the buffer. Allow the buffer to drain by gravity and discard.
3. Place the column in a collection tube
(Insert column into a 1000 ul pipet tip (cut the pipet tip off so that the column fits in the tube).
Place both of them into a 15 ml centrifuge tube, and centrifuge at 1100 x g for 2 minutes (2000 rpm) (centrifuge in swinging bucket centrifuge). Discard the pipet tip and the eluted buffer.
4. Place the column into a collection tube also inside the 15 ml centrifuge tube. Keeping the column in an upright position, very carefully apply the probed DNA sample (about 20 ul)(up to 100 μl) to the center of the column bed.
Note: Avoid applying the sample to the sides of the column; if this occurs, nucleotides flow around the medium and are not retained. Overloading the column (volume >100 μl) also results in nucleotides flowing through, contaminating the DNA sample.
5. Centrifuge for 4 minutes at 1100 x g (2000 rpm).
6. Save the eluate from the second collection tube.
This contains your purified DNA sample.
7. Discard the column into a designated radioactive waste container.
8. Test concentration in scintillator (LS 6500 Multi-Purpose Scintillation Counter. Pipet a 1 ul sample onto a Ready Cap with xtalscint (Beckman Coulter) in a standard 20ml liquid scintillation vial.
Or: Use Ecosinct H by National Diagnostics: pipet 5 ml into scintillation vial & pipet appropriate amount of probe (1ul, then adjust, **Best read is between 1mil-2mil cpm)
Machine Use
1) Choose Review & Edit User Prgms
2) Choose Cherenkov (w/o scintillate)
3) Find corresponding # (a black note) and insert number outwards in the middle of the rack (look for the “L” on the front).
4) Put the vials in the rack; put t he rack inside the machine; push “main menu,” then select “automatic count,” then push “start.”
5) To stop the machine, push the “reset” button.
9. Dilute probe to 100,000 cpm/ul
D. Preparation of non-denaturing acrylamide gel
Hoefer 16×18 cm 5% 1 gel: 30ml 2 gels: 60ml
Acrylamide (30%) 4 ml 8 mL
10X TBE 1.5 ml 3 mL
50% Glycerol 1.5 ml 3 ml
ddH2O 22.6 ml 45.2 mL
10% APS 300 ul 600 uL
TEMED 30 ul 60 uL
Hoefer 16×18 cm 5% 1 gel: 30ml 2 gels: 60ml
Acrylamide (30%) 5 ml 10 mL
10X TBE 1.5 ml 3 mL
50% Glycerol 1.5 ml 3 ml
ddH2O 21.6 ml 43.2 mL
10% APS 300 ul 600 uL
TEMED 30 ul 60 uL
Hoefer 16×18 cm 6% 1 gel: 30ml 2 gels: 60ml
Acrylamide (30%) 6 ml 12 mL
10X TBE 1.5 ml 3 mL
50% Glycerol 1.5 ml 3 ml
ddH2O 20.6 ml 41.2 mL
10% APS 300 ul 600 uL
TEMED 30 ul 60 uL
Hoefer 16×18 cm 7% 1 gel: 30ml 2 gels: 60ml
Acrylamide (30%) 7 ml 10 mL
10X TBE 1.5 ml 3 mL
50% Glycerol 1.5 ml 3 ml
ddH2O 19.6 ml 43.2 mL
10% APS 300 ul 600 uL
TEMED 30ul 60 uL
(For stat3 EMSA, 6% gel is recommended.) Leave the gel at least for 2 hour to polymerize. After removing the comb, use a pipet to clean the debris in each well. Then apply MQ water to rinse the well for three times. Pre-run the gel for at least 60 min and make sure the electric current no longer changes. It is better to also pre-run the gel at 4O C if you decide to run samples in the cold room.
At the beginning, using 80 V voltage to run the gel until all the samples get into the gel. Then voltage could be increased to 150-180 V. Stop running when the BPB reaches the bottom of the gel. The gel could be also run overnight by a very low voltage of 50 V. It will give a better picture.
E. Preparation of Loading Samples:
Nuclear Protein 5-10ug
10×BS Buffer 2ul
Delta&F 10ul
10mM DTT 2ul
Poly(dI-dC) 3ug
Probe (50kcpm/ul) 1-1.5ul
Final Total Volume 20ul
1.10xBS Buffer 10ml
50mM MgCl20.10165g
340mM KCl 0.25347g
Store in aliquots at -800C
2.δ & F 10ml
0.1mM EDTA 2λ 0.5M EDTA pH8
40mM KCl 400λ 1M KCl
25mM Hepes pH 7.6 (KOH) 250 λ 1M Hepes pH 7.6
1mM DTT( add prior to use) 100 λ 100mM DTT
8% Ficoll 400 0.8g Ficoll
Store in aliquots at -800C
3.poly(dI/dC)
1.5ug/ ul
Store in aliquots at -200C
Prepare all the constitution except probes, and then incubate on ice for 15-30 minutes. (If super-shift is necessary, pre-incubate the antibody with the mix for 30 minutes in room temperature.) Add probes to each sample and then incubate in room temperature for 30 minutes.
Do not load the sample with the loading dye/BPB. Load 5 ul 0.1% BPB in a separate well, that which is not adjacent to the other wells.
F. Drying gel and exposure
(1). Drying gel
1.Remove gel plates from apparatus & pry the glass plates off.( The gel
will stick on tone of a glass plate) Before detaching the gel, rinse the
gel with 100 ml fixing solution (10% acetic acid + 10% methanol) and
then with water. Remove excess water from the gel!
2.Put chromatography paper (Whatman Cat #3030 392) on top of gel and
lift up Whatman (the gel will miraculously come off the glass with the
Whatman!). Cover the other side of the gel with saran wrap (sandwich
the gel).
3.Gel Dryer: Remove plastic covers of the machine and place the
sandwiched gel on to the top of the dryer (Whatman on bottom)
4.Cover the gel with the two plastic covers. Make sure the plastic
covers are sealed tightly by pressing down on them.
5.Turn on the heater (set to 80o C & set timer at least 30 min). Turn on
the vacuum
6.Make sure the vacuum is sealed by pressing down on the plastic covers
(the plastic covers should be sucked in)
(2) Exposing the gel at -800C overnight using MS X-Ray film with the cassettes with intensifying screen.
Endnotes:
For liver regeneration following 70% partial hepatectomy, phosphatase inhibitors like NaF, NaMoO4, and Na3VO4 are not suggested. However, for liver regeneration following CCl4 treatment, all the phosphatase inhibitors are worth trying.