Overview

  • Product name

    Nuclear Extraction Kit
    See all Nuclear Extraction kits
  • Sample type

    Tissue, Adherent cells, Suspension cells
  • Assay time

    1h 00m
  • Product overview

    Nuclear Extraction Kit (ab113474) provides a simple and selective method along with all necessary reagents for nuclear protein extraction / nuclear protein fractionation in just 1 hour.


    The extracts can then be used in western blotting, protein-DNA binding assays, nuclear enzyme assays or any other procedures requiring optimized nuclear proteins. The protocol is fast and easy-to-use, and isolates very abundant yields of nuclear extract from mammalian cells or tissue samples.


    Not sure if this is the right product for you? Check out our EpiSeeker Sample Preparation Guide for help.


    Compared to other kits that use conventional nuclear extraction / nuclear fractionation methods, the buffers included in ab113474 contain much lower amounts of salts (80% less than conventional kits) and no SDS, which allows much better retention of enzyme activity in the nuclear extracts.

Properties

Images

  • Effect of two drugs refered to as Cilo (50 and 100 mg/kg; Cilo50and Cilo100), and Pio (3 and 10 mg/kg; Pio3 and Pio10), and their combination (Cilo50and Pio3) on the PPAR-γ transcription activity in rats subjected to ischemia (45 min)/reperfusion (24 hrs).
    Drugs were administered orally for 14 days then subjected to ischemia/reperfusion. Values are expressed as mean ± S.E.M (n = 6). Data are compared with sham operated control (#), I/R control (∗), Cilo50 (), Pio3 (‡), and combination (§) pretreated groups (one-way ANOVA followed by Tukey Multiple Comparison Test) at P<0.05.

  • B16F10 cells were treated with 30 µM of DMPB for the indicated time periods. Cytoplasmic and nuclear fractions were isolated and analyzed by Western blotting.
  • Nuclear extracts were prepared from MCF-7 cells and the activity of HDACs were measured using different amounts of the extract. The result shown in the figure demonstrates the ab113474's high specificity.

Protocols

References

This product has been referenced in:

  • Granata M  et al. S100A7, Jab1, and p27kip1 expression in psoriasis and S100A7 CRISPR-activated human keratinocyte cell line. J Cell Biochem 120:3384-3392 (2019). Read more (PubMed: 30203426) »
  • Srinivasan S  et al. SUMOylation of G9a regulates its function as an activator of myoblast proliferation. Cell Death Dis 10:250 (2019). Read more (PubMed: 30867409) »
See all 63 Publications for this product

Customer reviews and Q&As

21-30 of 33 Abreviews or Q&A

Answer



Are you sticking to the protocol steps of the booklet? Are you using cell culture samples?

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Answer


Unfortunately, we are not able to release the salt concentrations for this product as it is proprietary information.

We aim to provide as much information as possible to our customers, so I am sorry that this has not been possible on this occasion.
In addition, do not add Glycerol to the Nuclear Extract Preparation, as this may interfere with future experiments. Moreover, the freezing process is important to stop the endo- proteolysis of the sample.

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Answer

Thank you for contacting us.


Yes, the histone modification enzyme contained in the nuclear proteins extracted with this kit is usually active. However the nuclear proteins extracted with this kit may not be suitable for using in substrate analysis such as Km and specificity as the amount and purity of enzymes contained in the extracts probably are not sufficient


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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Answer

Thank you for contacting Abcam.

Please find the answers to your questions below:

1. Yes, the nuclear extracts prepared with this kit can keep the activity of these enzymes.

2. To study substrate specificity, the purified enzymes are much better than nuclear extracts because of low amount and less purity of enzymes in the nuclear extracts.

Please let me know if there is anything else I can help you with.

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Answer

Thank you for contacting us.
The lab let me know the following:
1. Both buffers ENE1 and ENE2 contain Triton X-100.
2. The buffers could be compatible with BioRad protein determination kits, especially with Bradford method.
3. The concentration of the 1x DTT is 1 mM.
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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Answer

Thank you for your inquiry.

I am happy to confirm, that we do have several such kits.The classical kit for nuclear protein extraction is the ab113474. https://www.abcam.com/index.html?datasheet=113474 (or use the following: https://www.abcam.com/index.html?datasheet=113474).I can however alsorecommend the kit ab113477 for example, which will allow extraction of all nuclear proteins in a nucleic acid free manner. Any enzymatic activity of a protein whould however been destroyed with this extraction kit. https://www.abcam.com/index.html?datasheet=113477 (or use the following: https://www.abcam.com/index.html?datasheet=113477).The nuclear protein extraction kit will also extract DNA binding proteins, as illustrated withthe grpah where HDAC has been detected. For histone specific extraction however, we recommend the ab113476 kit. Indeed, Histones do bind even more strongly to the DNAhttps://www.abcam.com/index.html?datasheet=113476 (or use the following: https://www.abcam.com/index.html?datasheet=113476).

I can stronglyrecommend you to look at our Guide, which will help you to choose the right kit for your application:
https://www.abcam.com/index.html?pageconfig=resource&rid=14321

I hope this information is helpful. Please do not hesitate to contact us again, should you have any further question.

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Answer

Thank you for your patience.

While we have not validated these extraction kits for use on the same samples, theoretically it should be fine. Whenusing the Plasma Membrane Protein Extraction Kit (ab65400), the nuclei will be present in thepellet that is discarded in step 4 of the Total Cellular Membrane section of our protocol. This pellet can, in theory, be saved and used in one of our nuclear or histone extraction kits.

I hope this helps, please let me know if you need any additional information.

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Answer

Thank you for contacting us.

I am checking for additional information about the compatibility of the plasma membrane protein extraction kit and a nuclear or histone extraction kit, but I do have information about some of your questions:

a) The Plasma Membrane Protein Extraction Kit (ab65400) is suitable for use on tissues as well as cells. When using tissues, simply skip steps 1 and 2 and proceed to 3: "For tissue samples, homogenize tissues in 2-3 volume of the 1X Homogenize Buffer, until it is completely lysed (30-50 times)..."

b) While both of the Episeeker extraction kits will be suitable for use on rat samples, and both will extract histones, the yield for histone proteins is much less using the Nuclear Extraction Kit (ab113474) than with the Histone Extraction Kit (ab113476).

I hope this helps. I will let you know as soon as I have additional information regarding your other questions.

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Answer

I apologize for the delay in my reply. If the protocol is followed closely and carefully, one should expect about 90% pure nuclear extract using ab113474. I hope this helps. Please contact me again if you have any further questions.

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Question

I have several questions about two products. EpiSeeker Nuclear Extraction Kit EpiSeeker Histone H3 (K27) Methyltransferase Activity Quantification Assay Kit 1. TheMethyltransferase Activity kit protocol has a note in the overview section that includes the phrase "HMT enzymes transfer a methyl group to histone H3 substrate from Adomet to methylate the substrate at lysine 4." Is this a typo? is the kit specific for K27 methyltransferase activity or will it also detect other methyltransferases? 2. There is another apparent typo in the Background section: "ab113454 has the following features: ...Quantitative determination of activity/inhibition of H3-K9 histone methyltransferases." 3. My samples for the transferase activity assay will be tissue extracts. One of the instructions in the nulcear protein extraction kit protocol recommends sonication: Nuclear Extract Preparation - "The extract (especially tissue extract) can be further sonicated for 3 X 10 sec to increase nuclear protein extraction." But could sonication diminish the methyltransferase activity in my samples? 4. The exrtraction kit protocol has all centrifuge spins specified in RPM. Can you provide the equivalent g forces? If not, can I assume the instructions apply to my microcentrifuge? What brand and model were used to develop the protocol? 5. I want to use the nuclear extracts for western blots, as well as the activity assay. Will the extracts be useful for blots of histones if I simply reduce and denature in the standard way for protein, given that most histone extraction protocols use an acid extraction? I am working with heterochromatin, and I am concerned that the extraction with this kit, which appears to be a low salt/high salt protocol, will notrelease the histones as efficiently as an acid extraction. 6. I have two concenrs about the DDT solution that is included with the extraction kit. Is it stable at 4C, or should this component be stored at -20C? My other concern is the appearance of precipitate, which does not go away when the 1000X stock is heated to 37C. I this normal?

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Answer

I received an update from the developer of the kit shortly after I sent my previous e-mail to you. The information they provided basically confirms what I had suggested in my e-mail, except that sonication will apparently not diminish activity: 1. Yes, these are both typos, they should be lysine 27. We apologize for any inconvenience caused by this. 2. 3 X 10 sec sonication at 4C will not affect enzyme activity. 3. It is about 11000 G (12000 rpm. Rotor radius = 7 cm for bench-top centrifuge such as Eppendorf 5415C). 4. The nuclear extract prepared with the kit is successfully used for WB histone detection. 5. The DTT solution is specifically formulated to be stored at 4C. It is stable at 4C for at least 6 months. Appearance of a very little precipitates is normal. It may be seen when storage temperature is <4C. Please contact me if you still have question or concerns.

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21-30 of 33 Abreviews or Q&A

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