Fine-tuning of Chromatin Immunoprecipitation (ChIP) for studying DNA-protein interactions in tomato

RICARDI, MARTINIANO MARÍA; GONZÁLEZ, RODRIGO MATÍAS; IUSEM, NORBERTO DANIEL

San Miguel de Tucumán, Tucumán.

Congreso; XLV Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2009

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Our goal is to develop a Chromatin Immunoprecipitation (“ChIP”) protocol suitable for tomato to search for DNA-protein interactions. For that purpose, we adjusted parameters to optimize the successive steps involved: Crosslinking (and its reversal afterwards): tissue was vacuum-infiltrated with different formaldehyde concentrations. The optimal one for achieving an efficient and reversible croslinking turned out to be 1%, as determined by phenol extraction (since only non- crosslinked DNA is recovered in the aqueous phase). Chromatin extraction: nuclei were purified by means of filtration steps through nylon mesh, centrifugation in buffers of different density and incubation with Triton detergent to lyse chloroplasts. Recovered nuclei were stained with Sybrgreen and observed in a confocal microscope as 5-10 mm fluorescent particles. DNA sonication: after testing various conditions, we ended up with 5 rounds of 10 seconds each at 15% amplitude, sufficient to obtain 200-1000 pb DNA fragments. Inmunoprecipitation: we performed overnight incubation trials with ChIP-grade anti-H3 antibody and non-immune serum as positive and negative controls, respectively. Real-time PCR: primers specific for actin exon 2 resulted in 0.997 amplification efficiency. Samples precipitated by anti-H3 antibody contained 139.4 (SD = 95; n = 4) times as much actin DNA as the negative control (SD = 0.35; n = 2).