Chromatin Extraction Kit (ab117152)

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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

  • Functional Studies - Chromatin Extraction kit (ab117152)
    Functional Studies - Chromatin Extraction kit (ab117152)
    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 Extraction Kit (ab117152)
    Chromatin Extraction Kit (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:
  • Fan P  et al. Overexpressed histone acetyltransferase 1 regulates cancer immunity by increasing programmed death-ligand 1 expression in pancreatic cancer. J Exp Clin Cancer Res 38:47 (2019). Read more (PubMed: 30709380) »
  • Zhu B  et al. The protective role of DOT1L in UV-induced melanomagenesis. Nat Commun 9:259 (2018). Read more (PubMed: 29343685) »
See all 6 Publications for this product

Publishing research using ab117152? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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1-10 of 22 Abreviews or Q&A

Question

Dear professor!

I konw the Nuclear Extraction Kit which productde by Abcam.We wander obtain the cell nucleus,and then extrat the nuclear DNA.We want to know whether the kit can do it?

Thanks very much!



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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.

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Question

I have a question regards to the chromatin extraction kit (ab117152). In the protocol for extraction using cell culture, it suggested that one lyse the cells by adding 200 uL/1 x 10^6 cells, while for the chromatin extraction buffer, one adds 50 uL/1 x 10^6 cells.

I have used this recommended volume, but my proteins in the chromatin fraction become a bit too diluted. And since I want to run them on SDS-PAGE, it would be a bit difficult to get up to 15 ug of protein into the wells (considering the excess dilution).

Therefore, I am writing to ask if you could advice me on how to extract chromatin extracted but not very diluted. For instance, would it be possible to use 100 uL/1 x 10^6 cells, instead of the 200 uL/1 x 10^6 cells? and can I use 25 uL/1 x 10^6 cells instead of the 50 uL/1 x 10^6 cells? This would be able to reduce the excess dilution for my sample.

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Answer

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.

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Question

I would like to use the chromatin extraction kit (ab117152) for protein work, to isolate the cytosolic from the chromatin fraction to probe for DNA binding proteins.

Hence, I would like to know whether the kit provides a pure fraction, that is, is the cytosolic fraction free from nuclear contamination, and is the chromatin extract clear from cytosolic contamination?

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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.

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Question

How much pure the chromatin fraction is?

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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.

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Question

I want to shear the chromatin, but rather than sonicate the sample I would like to use a micrococcal nuclease. This enzyme only works in the presence of Ca (reaction buffer: 50mM Tris.HCl pH 8, 5mM CaCl2), and here comes my question: does the Extraction Buffer of your kit contain calcium chelators (e.g. EDTA, EGTA)? If yes, could you give me some advice on how to use your kit in combination with the micrococcal nuclease (perhaps suggesting a self-made chromatin extraction buffer without chelators)?

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Answer

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.

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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

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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.

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Question

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 usiong 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?

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.

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Question


I am using Covaris ultrasound sonicator and will try to lyse the pellet as you suggested. My assumption is that after sonication I’ll pellet the debris and will try to see the chromatin on the gel so I can estimate the avarege size of DNA fragments. Then if I have good yield I can proceed with imunoprecipitation to obtain chromatin sample for Chip-chip assay.



Thank you for the suggestions and I’ll let you know if that method worked for obtaining chromatin DNA.

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Answer

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

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Question

I am using the Episeeker chromatin extraction kit and am seeing the genomic DNA in the pellet instead of the supernatent as expected. I am using frozen heart tissue. I suspect the tissue is not being lysed properly, and that this may be specific to heart tissue.Can you suggest anything to extract the chromatin, such as a different buffer or method?

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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.

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Question

I am using the Episeeker chromatin extraction kit and am seeing the genomic DNA in the pellet instead of the supernatent as expected. I am using frozen heart tissue. I suspect the tissue is not being lysed properly, and that this may be specific to heart tissue.Can you suggest anything to extract the chromatin, such as a different buffer or method?

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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!

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