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. Add 675 ul of 37% Formaldehyde stock directly to the culture medium to give a final concentration of 1%.  Incubate cells are room temperature for 10 minutes.

3. Dislodge cells from the T-flask, transfer to centrifuge tube on ice, and collect by centrifugation at 700 x g (SS34 – 3000rpm) for 10 minutes.

4. Wash cells with 10 ml cold PBS buffer.  Collect cells by centrifugation at 700 x g for 10 minutes.

5. Resuspend cell pellets in 300 ul of SDS lysis buffer (1% SDS; 10 mM EDTA; 50 mM Tris-HCl, pH 8.1), and agitated intermittently for 10 min on ice.

6. Transfer cell lysate to a 1.5 ml 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.

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

8. Dilute lysate 10 fold in IP buffer (0.01% SDS; 1.1% Triton X-100; 1.2 mM EDTA; 16.7 mM Tris-HCl, pH 8.1; 16.7 mM NaCl; 1mM PMSF; 1 ug/ml aprotinin; 1 ug/ml pepstatin A) and distribute evenly among three 1.5 ml tubes

9. Preclear the chromatin solution by adding a total of 80 ul of 50% protein A-sepharose beads (suspended in 10 mM Tris-HCl, pH; 1 mM EDTA; 1mg/ml BSA) and agitating the solution for 30 min at 4 °C. Beads were removed by centrifugation.

10. Use 300 ul of chromatin solution for each immunoprecipitation.  Add 1 to 5 ul of antiserum or control seum and incubate overnight at 4°C.

11. Add 35 ul of 50 % protein A sepharose, and incubate for 2 hr at 4 °C.

12. 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)

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

14. 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.

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

16. 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.

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

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