Overview

  • Product name
    Chromatin Extraction Kit
    See all Chromatin Extraction kits
  • Sample type
    Tissue, Adherent cells, Suspension cells
  • Assay time
    1h 00m
  • Product overview

    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.

     

Properties

Images

  • ChIP analysis of RNA polymerase II enriched in GAPDH and MLH1 promoters with chromatin extract prepared from formaldehyde fixed colon cancer cells (2x105) using ab117152.
  • Chromatin was extracted from 3x106 mouse neuroblastoma cells, fixed for 10min at RT in 0.1% formaldehyde. Cells were sonicated before 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

Protocols

References

This product has been referenced in:
  • Zhu B  et al. The protective role of DOT1L in UV-induced melanomagenesis. Nat Commun 9:259 (2018). Read more (PubMed: 29343685) »
  • Lu R  et al. COPS5 amplification and overexpression confers tamoxifen-resistance in ERa-positive breast cancer by degradation of NCoR. Nat Commun 7:12044 (2016). Functional Studies ; Human . Read more (PubMed: 27375289) »
See all 4 Publications for this product

Customer reviews and Q&As

1-10 of 22 Abreviews or Q&A

Answer

Thank you for your inquiry.

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

ab113474

https://www.abcam.com/index.html?datasheet=113474 (or use the following: https://www.abcam.com/index.html?datasheet=113474).

ab113477

https://www.abcam.com/index.html?datasheet=113477 (or use the following: https://www.abcam.com/index.html?datasheet=113477).

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/index.html?datasheet=117152 (or use the following: https://www.abcam.com/index.html?datasheet=117152).

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.

Read More

Answer


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.

Read More

Question
Answer

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.

Read More

Question

The Sonicator I use is Bioruptor UCD-200, a water bath based sonicator. I tried to use it so sonicate pure genomic DNA, by 10 cycles (30 sec on/30 sec off) it became 200 to 1000 bp size. When I sonicated chromatin, after 20 cycles, it became 1k to 3k. With more sonication up to 90 cycles, the size was still 1k to 3k. The size is based on the sonicated chromatin directly run in 1% agarose gel. Someone suggested that chromatin may migrate in the gel differently from pure DNA. I tried to purify DNA with Zymo’s DNA Clean and Concentrition column directly from sonicated chromatin or after treatment (boil, Proteinase K), but failed to get DNA back. The column works when I use pure DNA. Maybe the buffer of the chromatin interferes with DNA’s binding to the column. Now my question is, when you check the size of the chromatin after sonication, do you run it directly in agarose gel? If you purify the DNA at this step, what method do you use? Thanks! Yongzhi Geng > > > "http://www.w3.org/TR/html4/loose.dtd"> > > >     >     >     href="https://www.abcam.com/assets/css/campaign.css" type="text/css"> > > > > > body { > margin: 0px; > margin-top: 10px; > font-family: arial, verdana, tahoma; > background-color: #F5F5EE; > > > p{ > padding-top: 4px; > margin: 0px; > line-height: 18px; > > body table{ > font-size: 12px; > line-height: 100%; > > > a { > text-decoration: underline; > color: #004a91; > > a:visited { > color: #0096db; > > a:hover { > color: #0096db; > text-decoration: underline; > > .mainText { > margin-left: 0cm; > font-weight: normal; > margin-left: 10px; > > .tagLine { > margin-left: 0cm; > font-weight: normal; > margin-left: 10px; > color: #FFFFFF; > > .tagLine a { > margin-left: 0cm; > font-weight: normal; > text-decoration: none; > color: #FFFFFF; > > .tagLink { > margin-left: 0cm; > font-weight: normal; > text-decoration: none; > color: #FFFFFF; > font-weight: bold; > > .salutation{ > margin-left: 0cm; > padding-bottom: 10px; > font-weight: bold; > margin-left: 10px; > > > > > > bgcolor="#F5F5EE" align="center" margin="0" style="table-layout:fixed"> > > > > > > bgcolor="#FFFFFF" border="0" style="margin-top: 5px; border: 1px solid > #BBB;" align="center"> > > > > > > > > > > > > > > > > > > > href="https://www.abcam.com/index.html? utm_campaign=CRM&utm_source=Abcam.CRM&utm_medium=Email&utm_term=Header"> src="https://www.abcam.com/ps/cms/emails/CRM-Header.jpg" width="600" > height="141" border="0" alt="abcam: discover more" > > > > > > > > >normal; margin-left: 10px;">Dear Yongzhi >normal; margin-left: 10px;">    >>  > Thank you for > contacting us. > > >> I have had the > opportunity to discuss your question with the lab. 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 this > information is helpful to you. Please do not hesitate to contact us if you > need any more advice or information. > >   Help us improve our service. href="https://www.abcam.com/index.html? pageConfig=technicalSurvey&intCCEID=4649811">Rate > your experience with us today.   >  >Best regards, > Keith > > Keith Beadle > Scientific Support Specialist > Abcam Inc. > www.abcam.com > >  >   >  >Your original inquiry to Abcam: >>> Customer kindly contacted us as they have found that their chromotin > extract may not be breaking down fully with sonication. They ask if, after > sonicating, is DNA purification required to perform ChIP using this kit? If > so, how do we recommend purifying? Do we have any ideas as to why the DNA > is not breaking by sonication and are there any step that they may take to > rememdy this issue? >   > > >normal; margin-left: 10px;">[CCE4649811] > > SID:42 > > > >normal; margin-left: 10px; color: #FFFFFF;">Discover > more at abcam.com > > > > > > > > Yongzhi Geng 530-752-6715 Rm 6404A GBSF, Clinical Nutrition UC Davis

Read More
Answer

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.

Read More

Answer

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.

Read More

Answer

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.

Read More

Answer

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!

Read More

1-10 of 22 Abreviews or Q&A

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

Sign up