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

Chromatin was harvested from RAW246.7 cells or tissue samples using the Magnify ChIP system (Invitrogen). Cross-linked, sheared chromatin was used for immunoprecipitation with protein A/G dynabeads coupled with anti-Smad7, rabbit anti-acetyl-histone H4 Lys12 (H4K12Ac) IgG (EMD Millipore, Billerica, MA) or isotype control from a different species (rabbit). Precipitated DNA was reverse cross-linked and amplified by PCR using primers specific for IKK-β promoter (Table 1). Data were expressed as fold change after normalization for background levels.

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Manufacturer protocol

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