MAGnify™ Chromatin Immunoprecipitation System

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-The 1.25 M glycine must be at room temperature before use. -The Lysis Buffer must be at room temperature and fully resuspended before use. Vortex briefly to resuspend.	-For each ChIP reaction, use 10,000–300,000 cells or 0.167–5 mg of tissue. -1–10 μg of antibody is a typical starting range. - 10-minute crosslinking step using formaldehyde at a 1% final concentration. -Keep the cell lysate cooled on ice during sonication.

Upstream tips
-The 1.25 M glycine must be at room temperature before use. -The Lysis Buffer must be at room temperature and fully resuspended before use. Vortex briefly to resuspend.
Protocol tips
-For each ChIP reaction, use 10,000–300,000 cells or 0.167–5 mg of tissue. -1–10 μg of antibody is a typical starting range. - 10-minute crosslinking step using formaldehyde at a 1% final concentration. -Keep the cell lysate cooled on ice during sonication.

Publication protocol

ChIP was performed using the Magnify Chromatin Immunoprecipitation System (Invitrogen), according to the manufacturer’s instructions. Cells transfected with PC4-Flag or Flag empty vector, treated with or without TSA, were collected and washed with 1x PBS, and then fixed with 1% formaldehyde for 10 min. Chromatin was then prepared by sonication shearing, optimized according to the manufacturer’s instructions. ChIP was performed on sheared chromatin from approximately 2 × 105 cells, using IgG antibody as a negative control or antibodies recognizing Flag, TFIIB, Pol II, H3K9-Ac, or H3.3. Chromatin purified from an equivalent number of cells not subjected to the immunoprecipitation step was used as the input control. Real-time PCR was performed on DNA isolated from each of the ChIP reactions using primer pairs specific for LHR 5′-ACTGGGCACTGTCGCAGGTC-3′ (sense) and 5′-CATGGCCGGCGAACTGGGCT-3′ (anti-sense) [6]. The amplified region of the 189 bp LHR promoter contains the relevant proximal Sp1 response element. For ReChIP analyses, complexes resulting from the primary immunoprecipitation were eluted from protein A-agarose beads by incubation of samples with 5 mM dithiothreitol at 37°C for 20 min. Supernatants, containing protein complexes eluted with PC4-Flag or Flag (control), were recovered by centrifugation and diluted in ChIP dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl; pH 8.1). A second round of immunoprecipitation was performed with the specified antibodies, including those against H3 acetylated at various individual Lys residues or H3.3, using the Magnify Chromatin Immunoprecipitation System (Invitrogen). Realtime PCR was performed on DNA isolated from each of the ChIP reactions using a primer pair specific for LHR described above. The 2−dCt method was used to calculate relative enrichment values for each antibody with respect to input control.

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Manufacturer protocol

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