Chromatin IP Burakof Protocol

From Mswiki

PROTOCOL - ChIPs (Chromatin ImmunoPrecipitation assay)

The starting point is near confluent grown cells. At least Ixl07 cells is needed per sample.

- Add I/IO111 volume of buffered 11% formaldehyde solution (11% formaldehyde, 0.1 M NaCl, ImM EDTA pH8, 50mM HEPES pH 7.4) to the media and incubate at RT for 20min (with some light mixing).

-Remove all solution from the cells and add 4ml of PBS and harvest cells by scraping on ice and spin down.

- Resuspend cells in cold homogenisation buffer (lOmM Tris-HCl pH7.4, 15mM NaCl, 60mM KC1, ImM EDTA, 0.1% Nonidet P-40, 5% sucrose, Ix protease inhibitors) and subsequently transfer to a pre-chilled Dounce homogenizer (VWR) with pestle A and cells lyse by five-to-ten strokes (step is optional but removes some debris).

- Transfer sample to a 15-ml conical centrifuge tube and to the bottom of the tube add a l-2ml sucrose pad (lOmM Tris-HCl pH7.4, 15mM NaCl, 60mM KC1, 10% sucrose, Ix protease inhibitors) carefully. Nuclei are isolated by sedimentation through the pad (3200g, 20min, 4C). Carefully remove the supernatant.

- Resuspend in ChIPs buffer (4mL) (lOmM Tris-HCl pH7.4, lOOmM NaCl, 60mM KC1, ImM EDTA, Ix protease inhibitors and 0.1% NP-40).

- Shear nuclei by sonication by two 2min pulses on ice using a microtip on setting 3 (immerse the tip 5mm into the solution to maximize shearing), (freeze here if needed) Check sonication effectiveness by reversing X-links and treat with proteinase K.. ..check length distribution on 1% N-AGE. Size around 500bp is optimal for promoter analysis and for cloning purposes.

- Clear solution by an extensive high speed spin (1Omin 14000g) and subsequently continue with supernatant. Optional: pre-IP-clear solution by adding protein A/G-beads (60-lOOu.L) and incubate on nutator for 30 min at RT.

- Dilute the solution 1/5-1/10 in ChIPs buffer. Take out a samples for antibody precipitation (at least three samples are needed; ChIPs antibody, unrelated antibody and sample without antibody) and one 1/10 of a sample as input control.

- Incubate @RT on a nutator with antibodies of choice (typically 4-20u.l of concentrated purified ec-body) for at least 3h and then add 30u.L of Ultra-link Protein-A/G beads (Pierce) and continue for one additional hour @RT.

- Wash beads with ChIPs buffer (twice) and twice with Wash buffer (lOmM Tris-HCl pH7.4, 5OOmM NaCl, 60mM KC1, Ix protease inhibitors, 1% NP-40 and 0.01% SDS). Then wash once with Li-buffer (250 mM LiCl, lOmM Tris pH7.4, 1%NP-40, 1% deoxycholate) and wash once with TE pH8 and subsequently add lOOuL TE pH6.8/l% SDS to the beads and incubate for 20 min at 75C. Transfer supernatant to a fresh tube and add 300u.L TE pH8 with Proteinase K and incubate for Ih at 56C. Subsequently, incubate at 75 degrees for 6h to over-night to reverse X-links. Purify DNA by phenol/chloroform extraction containing 0.6M NaAc pH8 and ethanol precipitate.

- Amplify the region of interest by semi-quantative PCR and analyse by agarose electrophoresis.

NOTE: IT CAN BE IMPERATIVE TO USE HIGHER SDS CONC. IN THE WASHES (0.1% INSTEAD) BUT CAN LEAD TO LOSS OF ALL IG-BINDING FROM THE BEADS..... Steven Burakoff Laboratory (2000)