Nuclear Protein Isolation:

1) Complete media needed for lysis and nuclear extraction as follows:
Lysis Buffer: 15ml Buffer A + 45µl 1M DTT + 45.5µl 0.33M PMSF + 6µl 5mg/ml leupeptin + 150µl 2.1mg/ml Aprotinin
Nuclear Extract Buffer: 5ml Buffer C + 25µl 1M DTT + 15.5µl 0.33M PMSF
Store on ice. Good for < 2 hour.

2) At one minute remaining for cell stimulations, remove plates from incubator. Aspirate Sup., and precisely as time expires wash cells (in 6 well plate) with 1ml 4 C HEPES-Saline solution. Completely aspirate HEPES-saline solution. Immediately, add 400µl of complete buffer A (lysis buffer). Place plates on ice for 15 minutes.

NOTE: FOUND FOR ADHERANT MACROPHGES BEST NOT TO WASH CELLS. ASPIRATE MEDIA, AND LYSE. (100mm PLATE WITH 1000µl LYSIS BUFFER AND WELL OF 6WELL PLATE WITH 400µl LYSIS BUFFER.)

3) After 15 minutes on ice, rigorously scrape cells, collect liquid into 1.5ml microfuge tubes.

4.) Centrifuge 14,000 RPM for 45 seconds. Aspirate Sup. resuspend pellet in 50µl complete Buffer C (Nuclear Extract buffer). Incubate 4ºC for 30 minutes with mild agitation (we use end over end rocker).

5.) After 30 minutes (do not exceed 60 minutes), centrifuge tubes 14,000 rpm for 10 minutes at 4ºC. Aliquot sup. and store at -70ºC until needed. We find best results if used within 5 days.
Use one aliquot for protein concentration analysis. We use Bio-Rad Protein Assay.

6.) Pour 20cm polyacrylamide gels, in 0.5x TBE as follows:
For two 6% gel in 0.5x TBE: 6ml 5x TBE + 12ml 30% Acrylamide,0.8%Bis + 40ml dH2O + 900µl 10% APS + 12µl TEMED. After Polymerization, pre-run in 0.5x TBE buffer, at 15 mA constant current until needed.

End Labeling of Oligo. Probes and EMSA

7.) End label DNA oligo's using T4 kinase (Gibco):
17µl dH2O + 1.2µl Santa Cruz DNA oligo. (approx 10pMol)+ 6µl 5x Forward Reaction Buffer + 4µl 32P ATP. Mix, centrifuge a few seconds to collect.
Finally add 2µl T4 kinase, mix gently with pipet tip.
Label oligos for 1 hour at 37ºC.
8.) Nearing the end of the incubation, thaw nuclear extracts on wet ice.
9.) Prepare EMSA incubation buffer by heating one tube (995µl) to 37ºC to ensure that all salts are in solution. To this add 5µl of 1M DTT.
10.) For each reaction tube add the following:
5µl EMSA incubation buffer, 2-5µg Nuclear Extract protein, and 1µl 3.3µg/µl poly dI:dC, Qs. to 27 l with ddH2O.
Incubate for 15 minutes at room temp. to ensure non-specific DNA binding.
11.) Purify labeled oligo from the free 32P ATP by use of Qiagen's QIAquick Nucleotide removal columns, according to manufacturer indications, briefly:
Mix labeling solutions (30µl) with 300µl of Buffer PN.
Add solution to a QIAquick spin column in a collection tube.
Centrifuge 1 minute at 6,000 RPM
Place column into a new collection tube, discard used tube with radioactive flow through
Wash column with 500µl Buffer PE, centrifuge 1 min at 6000 RPM.
Discard flow through, repeat wash.
Discard flow through, centrifuge empty column 10,000xg for 1 minute
Place column in fresh microfuge tube, add 50µl dH2O, collect labeled
probe by centrifugation at 10,000xg for 1 minute.
12.) Aliquot 3 l (approx 100-200fMol ) labeled probe into each of the appropriate reaction tubes. Incubate 20 minutes at room temperature.
For competition, add 10x unlabeled competitor oligo.
For Supershift, add 1 l 2 g / l Ab. after 20 minutes incubation above, incubate additonal 20 minutes. WHEN DOING SUPERSHIFT, BEGIN THIS TUBE PRIOR TO OTHERS!!!

13.) Load 20µl of samples DO NOT USE loading dye. Run gel 15mA constant current for approximately 2 hours.
14.) Dry gel at 70ºC until dry (approx. 30 minutes).
15.) Autoradiograph and/or phosphorimage gel.

Solutions:

Lysis Buffer
0.2603g HEPES (10mM)
0.07455g KCl (10mM)
0.003802g EDTA (0.1mM)
0.625ml NP-40
100ml dH2O
Store 4 C until needed, see protocol to complete media.

Nuclear Extract Buffer
0.5206g HEPES (20mM)
2.4545g NaCl (0.42M)
0.01901g EDTA (5mM)
10ml Glycerol
90ml dH2O
Store 4 C until needed, see protocol to complete media.

5x TBE: 54g TRIS (445mM)
27.5g Boric Acid (890mM)
20ml 0.5M EDTA, pH 8.0 (10mM)
qs. to 1000ml dH2O

5x Incubation Buffer: 0.1901g EDTA (5mM)
2.92g NaCl (500mM)
0.788g TRIS"HCl (50mM)
500µl 1M stock MgCl2 (5mM)
20ml Glycerol
80ml dH2O
Mix well, aliquot 995µl/tube.
On experiment day, add 5µl 1M stock DTT (5mM)

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