Chromatin Extraction Kit (ab117152) 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. The isolated chromatin can also be used in other chromatin-related applications such as in vitro protein-DNA binding assays and nuclear enzyme assays.
Store at +4°C. Please refer to protocols.
|1000X Protease Inhibitor Cocktail||1 x 110µl|
|10X Lysis Buffer||1 x 11ml|
|Chromatin Buffer||1 x 11ml|
|Extraction Buffer||1 x 11ml|
ChIP analysis of RNA polymerase II enriched in GAPDH and MLH1 promoters with chromatin extract prepared from formaldehyde-fixed colon cancer cells (2×105) using ab117152.
Chromatin was extracted from 3×106 mouse neuroblastoma cells, fixed for 10min at RT in 0.1% formaldehyde. Cells were sonicated before the last centrifugation, and following centrifugation, cells were treated with proteinase K & RNAse A. Chromatin yield was 4 µg chromatin/106 cells. Lane 1: 1kb DNA ladder Lane 2: 300 – 650bp DNA fragments
Image courtesy of anonymous Abreview.
I can confirm that the chromatin fraction is 90% pure in general but it also depends on additional factors such as the cell/tissue type, and the chromatin extraction itself such as how effectively the cytoplasmic fraction is removed.
The extraction buffer of this kit contains a low concentration of EDTA which should not significantly affect the combination use of micrococcal nuclease with this kit as Ca++ concentration is 5 mM.
The size can be checked by directly running the sheared chromatin as seen in the attached file. If DNA will be purified. The following quick method may be useful: a) Use 10 uL sample and add 40 uL H2O b) Reverse cross-link by adding 2 uL of 5 M NaCl (Final concentration 0.2 M NaCl) c) Boil for 15 minutes d) After returning to room temperature, add 1 uL of 10 mg/ml RNase A at 37 °C for 10 mins e) Clean and purify DNA with PCR Clean-Up Kit, f) Load 1 and 4 uL of sonicated DNA on gel and determine the size of smear g).
The sonication condition that gives a smear of DNA sizes from 200 bp to 1 kb with a peak around 500 bp (2-3 nucleosomes) should be used for ChIP reactions. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.