The ChromaFlash™ Chromatin Extraction Kit is a complete set of optimized buffers and reagents for isolating chromatin or DNA-protein complex from mammalian cells or tissues in a simple and rapid format. Chromatin prepared by this kit can be used in a variety of chromatin immunoprecipitation methods. It is also the recommended method for obtaining chromatin required by Epigentek’s one-hour ChIP method using the ChromaFlash™ One-Step ChIP Kit. The isolated chromatin can also be used in other chromatin-related applications such as in vitro protein-DNA binding assays and nuclear enzyme assays.
- Extremely fast procedure: the entire procedure from cell/tissue sample to ready-to-use chromatin is less than 60 minutes.
- Convenient and flexible: the kit is suitable for preparing both native chromatin and cross-linked chromatin from monolayer or suspension cells, or from tissues.
- Unsheared chromatin makes it customizable for various analysis workflows that require either intact or fragmented chromatin, including ChIP, in vitro protein-DNA interaction analysis, nuclear enzyme assay, etc.
Chromatin immunoprecipitaton (ChIP) offers an advantageous tool for studying protein-DNA interaction. With ChIP, the experimenter can determine if a specific protein binds to the specific sequences of a gene in living cells by combining with PCR (ChIP-PCR), microarray (ChIP-chip), or sequencing (ChIP-Seq) techniques. For example, the measurement of the amount of methylated histone H3 at lysine 9 (meH3-K9) associated with a specific gene promoter region under various conditions can be achieved through a ChIP-PCR assay, while recruitment of meH3-K9 to the promoters on a genome-wide scale can be detected by ChIP-chip. In particular, the ChIP method with specific antibodies directly against various transcriptional factors is widely demanded.
Principle & Procedure
The ChromaFlash™ Chromatin Extraction Kit contains all reagents required for carrying out successful chromatin extraction directly from mammalian cells or tissues. Cell membranes of the sample, with or without cross-linking, are broken down using the provided lysis buffer. Chromatin or DNA-protein complex is then extracted with the extraction buffer. The extracted chromatin can then be diluted with chromatin buffer and stored at the appropriate temperature.
Starting Materials & Input Amount
Starting materials can include various tissue or cell samples such as cells from flask or microplate cultured cells, fresh and frozen tissues, etc. The amount of cells and tissues for each preparation can be 1 x 105 to 5 x 106 cells and 10 mg to 200 mg, respectively. For optimal preparation, the input amount should be 1 to 5 x 106 cells or 50 to 200 mg tissues. Yield of chromatin is approximately 4 ug per 106 cells or per 50 mg tissues.
Fig. 1. Schematic procedure of chromation isolation with the ChromaFlash™ Chromatin Extraction Kit.
Fig. 2. ChIP analysis of RNA polymerase II enriched in GAPDH and MLH1 promoters with chromatin extract prepared from formaldehyde fixed colon cancer cells (2x105) using the ChromaFlash™ Chromatin Extraction Kit.