Chromatin Extraction Kit (ab117152)

Thank you for your inquiry.

We have two Nuclear Extraction Kits in our catalog at the moment:

ab113474

https://www.abcam.com/Nuclear-Extraction-Kit-ab113474.html (or use the following: https://www.abcam.com/Nuclear-Extraction-Kit-ab113474.html).

ab113477

https://www.abcam.com/Nuclear-Nucleic-Acid-Free-Extraction-Kit-ab113477.html (or use the following: https://www.abcam.com/Nuclear-Nucleic-Acid-Free-Extraction-Kit-ab113477.html).

Both kits are intended for the extraction of protein from the nucleus.

ab113477 is designed to extract Proteins nucleic acid free.

We also have ab117152 (EpiSeeker Chromatin Extraction Kit) that will extract DNA and theDNA bound chromatin.

https://www.abcam.com/Chromatin-Extraction-Kit-ab117152.html (or use the following: https://www.abcam.com/Chromatin-Extraction-Kit-ab117152.html).

I am sorry to confirm that we do not have kits specifically for protein free DNAextraction.

I would also like to make you aware that we do have scientific support in Chinese. If you prefer to speak to us in Chinese please write to mailto:hk.technical@abcam.com.

Under these conditions it should be fine to reduce the amount of working extraction buffer so that the chromatin is less dilute however sonication using a water bath sonicator may be necessary to improve rupture of the nuclear membrane and release of chromatin material. The recommended amount of Working Lysis Buffer may still be used since the supernatant from the lysis is removed after centrifugation.

In general the chromatin fraction is 90% pure but it also depends on additional factors such as the cell/tissue type, and the chromatin extraction procedure itself such as how effectively the cytoplasmic fraction is removed.

Likewise, the cytosolic fraction is generally free from nuclear contamination and is about 90% pure.

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 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 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.

In general, the chromatin is easy to be sonicated to 200-1000 bps. If the chromatin extracts can not be broken down fully by sonication, it may be due to insufficient time length, power intensity, foaming of chromatin solution during sonication etc. these sonicatio parameters may need to be optimized based on the sonicator type before ChIP. for the best sonication results, we recommend to use water-bath-based sonicators such as Episonic 1100 or covaris, with the established chromatin shearing protocol. DNA purification is not required before ChIP. Cleaned up and eluted DNA after ChIP can be directly used for PCR. However for microarray or sequencing use, the DNA should be further purified as indicated in the user guide.

I hope the sonication step is helpful. I look forward to hearing how things go!

In general, this kit could generate about 4 ug/1 million cells of chromatin or 2 ug of DNA (histone:DNA in chromatin is about 1.8-2 :1). Based on the described conditions by the customer, the relatively low yield of DNA seems mainly due to that nucleus are not lysed or broken completely although the cell type may also affect the DNA yield.

To increase DNA yield,I recommend treatingthe pellet as follows:

Resuspend and sonicate 2 X 20 seconds to increase chromain extraction. Allow the sample to cool on ice between sonication pulses for 30 seconds. As an example, sonication can becarried out with a microtip attached to Branson 450 sonifier, setting at 25% power output.

I hope this is helpful. I look forward to hearing if this improves your results.

Thank you for your enquiry. My colleague Karen asked me to follow up with you.

How has it been determined that the chromatin is in the pellet rather than the supernatant?

Thanks in advance for the additional information!