Chromatin immunoprecipitation assay with urea denaturation (see step 9). (This is based on Chanhyo’s protocol which in turn is an amalgam of Wu et al. 2003, Park et al. 2001 and the Upstate Biotechnology ChIP assay kit.  This is our preferred procedure because the signals are about 10 times stronger than the Upstate Biotechnology procedure.)

1. Grow 25 ml of Drosophila S2 cells in a T-flask at 25°C in Schneider’s Drosophila medium (Gibco BRL) supplemented with 10% heat inactivated fetal bovine serum to a density of 4~5x106 cells/ml.  (If desired, heat shock the cells by placing the T-flask in a 37°C water bath for 15 minutes.  Support the T-flask horizontally in the water bath so the cells don't become anoxic.  Cool back back to room temperature in a water bath for several minutes before adding formaldehyde.)

2. Formaldehyde crosslinking:

Add to the cells, 2.5 ml of 11% formaldehyde solution (prepared from 37% formaldehyde, 10% methanol stock solution from Merck) in 0.1M NaCl, 1mM EDTA, 0.5mM EGTA, 50mM Tris-HCl (pH 8) to the final concentration of 1%. (Orlando and Paro, 1993; Park et al, 2001).  Incubate at room temperature for 10 minutes. 

3. Quench the crosslinking by adding 2M glycine to a final concentration of 240 mM (Komarnitsky et al, 2000).

4. Collect cells by centrifugation at 700 x g (SS34 – 3000rpm) for 10 minutes.

5. Resuspend and wash cells with 10 ml cold PBS buffer.  Collect cells by centrifugation at 700 x g for 10 minutes.

6. Resuspend cell pellets in 1 ml of sonication buffer {10mM Tris-HCl pH 8.0, 1mM EDTA, 0.5mM EGTA (Park et al, 2001), supplemented with fresh protease inhibitors as follows: 1ug/ml aprotinin, 1ug/ml pepstatin A, 0.5mM PMSF (Boehm et al, 2003)}.

7. Transfer cell suspension to a 1.5 ml microfuge tube.  Mount tube in an ice/salt bath with the sonicator.  Sonicate 30 cycles: 10 sec. on, 30 sec. off.  Average length of the DNA should be 500 bp.  This can be checked by taking 5 ul of solution, treating with RNase A for 30 min., and running on a gel.

8. Centrifuge at 13,000 rpm at 4 °C for 10 min to remove debris.

9. Transfer supernatant to a 15 ml tube, and mix with an equal amount of 6M urea. (Park et al, 2001; Boehm et al, 2003: this is a significant departure from most protocols but improves signals by as much as 10 fold.)

10. Dialyze overnight in the cold against 200 ml of ChIP buffer (10mM Tris-HCl pH 8.0, 1mM EDTA, 0.5mM EGTA, 0.5mM PMSF, 10% glycerol, 0.1% Na-deoxycholate, 1.0% Triton X-100) (Park et al, 2001; Boehm et al, 2003)

11. Centrifuge to remove debris. Transfer chromatin solution to new tube. It should be around 1.5 ml. (Chromatin solution can be stored at -70°C, but don’t forget the subsequent preclearing step.)

12. Preclear the chromatin solution by adding 80 ul of 50% protein A sepharose beads (suspended in 10 mM Tris-HCl pH 8, 1 mM EDTA, 1 mg/ml BSA and transferred with a pipette whose tip is cut to widen the hole) and mix the solution gently for 30 min at 4 °C. Centrifuge at 13,000 rpm for 5min to remove beads. Transfer supernatants to fresh siliconized tube. (Chromatin solution can be stored in -70°C for future use; it does not require another preclearing although it should be centrifuged to remove insoluble debris.)

* Preparation of 50% protein A sepharose:  Put 100 ul of dry beads in a 2 ml screw cap tube and swell with 1.5 ml of water for at least 15 minutes on a rotator at 4°C.  Collect beads at 2000 rpms for 3’ and discard supernatant.  Resuspend beads in 1.5 ml of water, mix for 5’, and recover beads.  Wash beads 2 times with 1.5 ml of TE.  Estimate the pack volume of beads and add 0.8 volumes of TE and 0.2 volumes of 10 mg/ml acetylated BSA (final BSA concentration should be 1 mg/ml).

13. Determine DNA concentration of chromatin solution using the fluorometer. Around 150 ul of chromatin solution (Calculate the amount of chromatin solution for one I.P. 5~10ug of DNA or 1 X 107 cells is used for one I.P.) will be used for I.P.

14.  Add antibody (4 ul of antiserum against GAGA, DSIF, TBP, TAF250, HSF, and Pol II and 7 ul of antiserum against NELF-B, D and E) to solution and incubate overnight at 4°C. (DSG: smaller amounts might work as these amounts were determined arbitrarily to give good results.)

15. Add 35 ul of 50 % protein A sepharose, and incubate for 2 hr at 4 °C. (DSG: An earlier protocol called for 70 ul which is excessive. A ratio of 5 ul of 50% protein A:1 ul of antiserum should suffice since the binding capacity of protein A sepharose is 20 ug of antibody per 1 ul of protein A separose.)

16. Collect the protein A sepharose beads by brief centrifugation at 3000 rpm for 5 minutes. (Store the supernatants at -70°C in case they are needed in the future)

17. Wash the immunoprecipitates (incubate each wash solution containing the immunoprecipitates at 4 °C for at least 5 minutes and centrifuge at 3000rpm for 5 min.)

(1) 6 times with 1 ml of low salt wash buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 150mM NaCl),

(2) 3 times with 1 ml of high salt wash buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 500mM NaCl),

(3) 2 times with 1 ml of Lithium Wash Buffer (0.25M LiCl, 1% NP-40, 1% sodium deoxycholate, 1mM EDTA, 10mM Tris-HCl pH 8.1), {The length of these washes can range from 10 minutes to leaving one overnight.  An overnight wash can significantly reduce background.  The best way to judge is to see how much background shows up in the control lanes.}

(4) once with 1 ml of TE buffer,

(5) resuspend in 1 ml of TE buffer and transfer to a fresh tube to eliminate DNA bound nonspecifically to the original tube’s surface.

18. Elute the protein–DNA complexes by adding 250 ul of Elution Buffer (1% SDS, 0.1M NaHCO3) and incubating the solution at room temperature for 15 min. Spin-down the beads at 3000 rpm and transfer the supernatants to new tubes. Elute once again with 250 ul of Elution buffer. In addition, prepare input DNA by adding Elution Buffer to 20% input to make final volume 500 ul.

19. Add 20 ul of 5M NaCl to the eluates and incubate at 65°C for 4 hr to reverse formaldehyde crosslinks.

20. Add 10 ul of 0.5 M EDTA, 20 ul of 1M Tris-Cl (pH 6.5), and 2 ul of 10 mg/ml proteinase K and incubate at 45 °C for 1 hr.

21. Phenol/chloroform extract 2 times, ether extract 1 time and evaporate off excess ether.  Add 20ug of linear polyacrylamide, 50 ul of 3M NaOAc, and 1 ml 100% EtOH.  EtOH precipitate as usual.

22. Dissolve DNA pellets in 30 ul of TE for PCR reactions and store at -70°C.

PCR for ChIP
water 38.1ul
Immuoprecipitated DNA 1ul
10 pmol/ul (0.01mM) primer 1 1ul
10 pmol/ul (0.01mM) primer 2 1ul
10 pmol/ul (0.01mM) primer 3 1ul
10 pmol/ul (0.01mM) primer 4 1ul
10mM dNTP mixture 1ul
10X Taq buffer (Gene Choice) 5ul
32P-dCTP 0.4ul (4uCi)
5U/ul Taq Polymerase 0.5ul
Total 50ul

Run 25 cycles.
Incubate samples at 94°C for 3 min (denaturation).
(1) Denaturation : 94°C, 1min
(2) Annealing : 55sec
(3) Extension : 72°C, 50sec
Incubate samples at 72°C for 10 min (final extension).

Run 10 ul of each PCR reaction with 2ul of 6X dye (0.25% bromophenol blue, 0.25% xylene cyanol FF, 15% ficoll (Type 400, Pharmacia, in water) on a 6% non-denaturing acrylamide in 1X TBE at 150V until bromophenol bule migrates out of the gel.

* Preparation of 6% acrylamide gel
5X TBE 4ml
30% acrylamide (29.2% acrylamide, 0.8% bisacrylamide) 4ml
Water 12ml
10% APS (ammonium persulfate) 140ul
TEMED (tetramethylethylenediamine) 15ul

Fix the gel in 10% acetic acid for 10~20 minutes and then dry. Detect the radioactivity in the dried gel using a phosphoimager (Molecular Dynamics). Quantify the intensity of each band by volume analysis using ImageQuant software. (The accuracy of the assay can be assessed by determining how well the measurements for inputs matched the expected ratio of 0.1:1:10. )