1.  Crosslink protein DNA complexes in vivo

            Grow cells in suspension and collect 5 x 10⁸ cells by low speed centrifugation.  Resuspend cells in 50ml media.  In fume hood, add 1.4ml 37% fomaldehyde solution to a 50ml conical tube.  Fill tube with cell culture to just below the 50ml line.  Incubate at room temperature for 15min. with occasional inversion.  (The extent of cross-linking is critical and depends on the protein of interest.  Too much cross-linking may mask epitopes and too little cross-linking may lead to incomplete fixation.  The concentration of formaldehyde, the length of cross-linking or the temperature of cross-linking can all be adjusted.)

 2.  Quench cross-links

            Add 3.4ml 2M glycine to fixed culture and incubate at room temperature for 5min. with occasional inversion.

 3.  Harvest cells

            Centrifuge cells (5min. at 3000rpm) and discard supernatant.  Wash cells with 10ml ice-cold 1X TBS and spin cells down, again and discard supernatant.  Place cells on ice.

 (Cells can keep on ice for a few hours, if you are collecting many samples for a time course.  Alternatively, cells may be frozen in liquid nitrogen and placed at -80°C).

 4  Lyse cells

            Resuspend cell pellet gently with a pipette in 10ml RIPA buffer with protease inhibitors and incubate on ice for 30min.  Further disrupt cells by passing them through a 21 guage needle.  Add 100ml 10mg/ml PMSF and incubate on ice for another 30min.  Transfer lysate to 2ml microcentrifuge tubes (1ml lysate/tube).

 5.  Shear chromatin

            Using a Branson 350 Sonifier with a microtip at a power setting of 7 and a 60% duty cycle, sonicate extracts for nine 10sec pulses.  In between 10sec. pulses, let samples sit on ice for atleast 2min.  This should shear chromatin to a final average size of 500bp.  (Your sonicator will have to be calibrated to yield the desired final average length of DNA).

 6.  Clarify samples

            Centrifuge samples at max speed for 5min at 4°C.  Transfer supernatant to a fresh 1.5ml microcentrifuge tube and centifuge samples again for 15min at max speed at 4°C.

 7.  Preclear extracts

            Add 30ml bed volume of Protein A sepharose beads to each tube and incubate on a rotation wheel for 50min. at 4°C.  Centifuge samples at 7500rpm for 2min and then transfer supernatant to a fresh tube.

 8.  Immunoprecipitation

            Add the primary antibody against the protein of interest to the extract.  (Preliminary immunoprecipitation experiments should be performed to determine the appropriate amount of antibody to be used).  Incubate on ice for 3hrs, then add 30ml bed volume Protein A sepharose beads.  Incubate on rotating wheel for 1hr at 4°C.  Centrifuge sample for 2min at 7500rpm at 4°C.  Keep 50ml of sample for sizing DNA and add 200ml 1%SDS/1X TE to it, discard the rest of the supernatant.

 9.  Wash immunoprecipitates

            Add 1ml RIPA buffer to the beads and incubate for 5min. on rotating wheel at 4°C and then centrifuge at 7500rpm for 2min.  Discard supernatant.

            Add 1ml RIPA-500 to the beads and repeat incubation and centrifugation.

            Add 1ml LiCl/detergent solution to the beads and repeat incubation and centrifugation.

            Add 1ml 1XTBS to the beads and repeat incubation and centrifugation.

10.  Elute immunoprecipitates

            Add 100ml 1%SDS/1X TE, mix and incubate at 65°C for 10min.  Centifuge briefly and transfer eluate to fresh tube and wash beads with 150ml 0.67%SDS/1X TE.  Briefly centrifuge and add wash to eluate.

 11.  Reverse cross-links

            Incubate the immunoprecipitates and the total extract material for at least 6hrs at 65°C.

 12.  Proteinase K treatment

            Add 250ml Proteinase K solution and incubate for 2hrs at 37°C.

 13.  Purify DNA

            Add 55ml 4M LiCl andd 500ml 25:24:1 phenol/chloroform/isoamyl alcohol.  Vortex vigorously for 1min.  Separate phases by centrifugation at max speed for 10min. at room temperature.  Transfer aqueous phase to a fresh tube and add 1ml 100% ethanol.  Mix and cetrifuge at max speed for 15min. at room temperature.  Discard the supernatant and dry pellet.  Resuspend DNA in 10ml 1XTE and store at -20°C

Analyze data by PCR assay or microarray analysis.

 Solutions:

 1X TBS

            150mM NaCl

            20mM Tris-HCl, pH7.6

 RIPA Buffer

            10mM Tris-HCL, pH8

            140mM NaCl

            0.025%NaN₃

                _{1% Triton X-100}

                _{0.1% SDS}

                _{1% Deoxycholic acid}

 Lysis Buffer-500

            10mM Tris-HCL, pH8

            500mM NaCl

            0.025%NaN₃

                _{1% Triton X-100}

                _{0.1% SDS}

                _{1% Deoxycholic acid}

LiCl/detergent wash

            0.5% Deoxycholic acid

            1mM EDTA

            250mM LiCl

            0.5% NP-50

            10mM Tris-HCl, pH8

Proteinase K solution

            1ml 20ml/ml glycogen

            5ml 20mg/ml Proteinase K

            244.5ml 1X TE, pH7.6