The ChromaFlash™ Chromatin Isolation & Shearing 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, followed by a shearing step by sonication. 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.
When performing ChIP, chromatin or DNA-protein complex in cells or tissues should first be isolated and sheared to a desired size. The ChromaFlash™ Chromatin Isolation & Shearing Kit addresses the inconvenience and time consuming issues of existing chromatin preparation methods by introducing the following features:
- Fast procedure: the entire procedure from cell/tissue sample to ready-to-use sheared chromatin is less than 2 hours.
- Convenient and flexible: the kit is suitable for preparing both native chromatin and cross-linked chromatin from monolayer or suspension cells, or from tissues.
- Chromatin shearing step is pre-optimized for sonication with various sonication platforms including waterbath-based (EpiSonic, Covaris, Bioruptor) sonicators, probe-based sonicators (Branson, Misonix), and enzyme-based methods.
Chromatin immunoprecipitaton (ChIP) offers an advantageous tool for studying protein-DNA interaction. The experimenter can determine if a specific protein binds to the specific sequences of a gene in living cells by combining ChIP 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, a ChIP method with specific antibodies directly against various transcriptional factors is widely demanded.
Principle & Procedure
The ChromaFlash™ Chromatin Isolation & Shearing Kit contains all reagents required for carrying out successful chromatin extraction and shearing 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 is then sheared according to the protocol's recommended protocol settings for a variety of sonicators of the experimenter's choice and stored at an appropriate temperature after being diluted with chromatin buffer.
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 2 x 105 to 1 x 107 cells and 10 mg to 400 mg, respectively. For optimal preparation, the input amount should be 2 to 5 x 106 cells or 100 to 200 mg tissues. A total of 100 standard extractions (using 2 x 106 cells or 100 mg of tissue per extraction) can be performed with this kit. The yield of sheared chromatin is approximately 3 µg per 106 cells or per 50 mg of tissue.
Fig. 1. Schematic procedure of the ChromaFlash™ Chromatin Isolation & Shearing Kit.
Fig. 2. Chromatin was extracted from six MDA-231 cell samples. 40 ul (8 ug) of chromatin were sheared in a 0.2 ml tube using the EpiSonic 1100 for 20 cycles (15 sec On and 30 sec Off). DNA was purified and then run on a gel.
Fig. 3. The sheared chromatin was used for ChIP-qPCR analysis of RNA polymerase II enrichment in GAPDH and MLH1 promoters by using the ChromaFlash™ One-Step ChIP Kit (P-2025) and the Methylamp™ MS-qPCR Fast Kit (P-1028).