EpiQuik Chromatin Immunoprecipitation (ChIP) Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Ensure that all buffers are in clear solution. Shake or vortex if these buffers precipitate.	-When processing spin columns, always cap spin columns before placing them in the microcentrifuge. -The conditions of crosslinked DNA shearing can be optimized based on cells and sonicator equipment. If desired, remove 5 µl of sonicated cell lysate for agarose gel analysis. The length of sheared DNA should be between 200-1000 bp.

Upstream tips
-Ensure that all buffers are in clear solution. Shake or vortex if these buffers precipitate.
Protocol tips
-When processing spin columns, always cap spin columns before placing them in the microcentrifuge. -The conditions of crosslinked DNA shearing can be optimized based on cells and sonicator equipment. If desired, remove 5 µl of sonicated cell lysate for agarose gel analysis. The length of sheared DNA should be between 200-1000 bp.

Publication protocol

ChIP was performed by using the EpiQuik Tissue Chromatin Immunoprecipitation Kit (Epigentek, Brooklyn, NY) (20). Briefly, mouse lung tissue was formaldehyde fixed and in vivo cross-linked, followed by tissue disaggregation. The cells were lysed, and the nucleoprotein complexes were sonicated to reduce the sizes of DNA fragments to 1,000–200 bp. Normal mouse IgG was used as the negative control and genomic and input chromatin as positive controls. Acetylated histone H3 and H4 antibodies (Millipore, Billerica, MA) were used for each immunoprecipitation. The immunoprecipitated DNA was eluted in a total volume of 20 μl, and 4 μl were used for PCR that was performed using a PTC-200 DNA engine (M. J. Research, Waltham, MA) under the following conditions: 94°C for 3 min; 32 cycles at 94°C for 45 s, 60°C for 1 min, and 72°C for 1 min; and final elongation at 72°C for 10 min. The following primer was used in PCR: IL-6, 5′-GAC ATG CTC AAG TGC TGA GTC AC-3′ (sense) and 5′-AGA TTG CAC AAT GTG ACG TCG-3′ (antisense), and PCR products were analyzed on a 1.8% agarose gel.

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Manufacturer protocol

Download the product protocol from Epigentek for EpiQuik Chromatin Immunoprecipitation (ChIP) Kit below.

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