ChIP Rat - Heart

DNA-protein interactions are studied by using ChIP. The basic steps in this technique are crosslinking, sonication, immunoprecipitation, and analysis of the immunoprecipitated DNA. During ChIP, if chromatin is under-fragmented or fragments are too large which can lead to the increased background and lower resolution. Shorter cross-linking times (5-10 min) and/or lower formaldehyde concentrations (<1%) may improve shearing efficiency. If Chromatin is over-fragmented, then optimize shearing conditions for each cell type to improve ChIP efficiency. Over-sonication of chromatin may disrupt chromatin integrity and denature antibody epitopes. If you do not see any product or very little product in the input PCR reactions, add 5–10 μg chromatin per IP.

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Found 3 matching solutions for this experiment

MAGnify™ Chromatin Immunoprecipitation System

Thermo Fisher Scientific

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.

Manufacturer protocol Publication protocol 1 Relevant paper

ChromaFlash Chromatin Extraction Kit

Epigentek

Upstream tips	Protocol tips	Downstream tips
- Spin the solution down to the bottom prior to use. - Check if any buffers contain salt precipitates before use. If so, shake the buffer until the salts are re-dissolved.	-For optimal preparation, the input amount should be 1 to 5 x 106 cells or 50 to 200 mg tissues. -Avoid multiple freeze/thaw cycles.

Upstream tips
- Spin the solution down to the bottom prior to use. - Check if any buffers contain salt precipitates before use. If so, shake the buffer until the salts are re-dissolved.
Protocol tips
-For optimal preparation, the input amount should be 1 to 5 x 106 cells or 50 to 200 mg tissues. -Avoid multiple freeze/thaw cycles.

Manufacturer protocol Publication protocol 1 Relevant paper

ChIP-IT® Express Chromatin Immunoprecipitation Kits

Active Motif

Upstream tips	Protocol tips	Downstream tips
-Do not re-freeze the Protein G magnetic beads, the beads should be stored at 4C.	-Always use ChIP validated antibody. -Optimal use of 1-3 ug of antibody.	-The diluted Proteinase K stop solution can not be stored.

Upstream tips
-Do not re-freeze the Protein G magnetic beads, the beads should be stored at 4C.
Protocol tips
-Always use ChIP validated antibody. -Optimal use of 1-3 ug of antibody.
Downstream tips
-The diluted Proteinase K stop solution can not be stored.

Manufacturer protocol Publication protocol 1 Relevant paper

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