Histone H3 (K9) Methyltransferase Activity Quantification Assay Kit (ab113453)

Reviews (1)Q&A (5)

Overview

  • Product name

    Histone H3 (K9) Methyltransferase Activity Quantification Assay Kit
    See all Histone Methyltransferase kits

  • Sample type

    Tissue Extracts, Cell Lysate

  • Assay type

    Quantitative

  • Assay time

    3h 00m

  • Species reactivity

    Reacts with: Mouse, Human
    Predicted to work with: Mammals

  • Product overview

    Methylation of histone H3 at lysine 9 (K9) mediates heterochromatin formation by forming a binding site for HP1 and also participates in silencing expression at euchromatic sites. In mammalian cells, histone lysine methyltransferases such as SuVar39h and ESET among others are responsible for methylation of lysine 9 at histone H3.

    Histone H3 (K9) Methyltransferase Activity Quantification Assay Kit (ab113453) allows the user to measure the activity or inhibition of individual histone methyltransferase (HMT) that specifically target histone H3 at lysine 9 (H3K9). The kit is ready-to-use and provides all the essential components needed to carry out a successful HMT activity/inhibition quantification experiment without the need for radioactivity or any special equipment.

    ab113453 recognizes methylated H3 at lysine site 9.

  • Platform

    Microplate reader

Properties

Images

  • Functional Studies - Histone H3 (K9) Methyltransferase Activity Quantification Assay Kit (ab113453)
    Functional Studies - Histone H3 (K9) Methyltransferase Activity Quantification Assay Kit (ab113453)
    Nuclear extracts were prepared from MCF-7 cells using the ab113474 and H3-K9 specific histone methyltrasferase activity (G9a) was measured. Purified G9a was spiked into the nuclear extract that has low concentration of nuclear proteins by dilution.

Protocols

References

ab113453 has not yet been referenced specifically in any publications.

Publishing research using ab113453? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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1-6 of 6 Abreviews or Q&A

Question

Questions to assay principle and componens:

1. Is the substrate a peptide or protein fragment?
2. Why is the substrate biotinylated? Is the plate avidin-coated? Or is other coating material used?
3. Which methylation is being detected by the capture antibody: mono, di, or tri-methyl or all of them?
4. Can different HMTs be differentiated within one sample?
5. Is the detection antibody HRP conjugated?
6. Which enzyme is being used as HMT standard?
7. Which enzyme is being used as control enzyme?
8. What is in the histone assay buffer, EHM2?

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Answer

Thank you for contacting us.
Here are the answers to your questions:
1. Peptide.
2. Yes, avidin-coated.
3. This information cannot be disclosed currently.
4. No. unless a purified enzyme or extract with the over-expressed specific enzyme are used.
5. Yes.
6. The standard is methylated H3K9 peptide
7. G9a
8. salt buffer with protein stabilizers
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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Standard Curve (plot of OD value versus amount of HMT Standard) for H3K9 Methyltransferase Activity Quantification Assay Kit.

 Excellent Good 4/5 (Ease of Use)
Abreviews
abreview image
A standard curve was plotted by plotting the blank corrected OD450 for three different HMT standard concentrations. This kit works well and is specific for H3K9 Me-transferase.

Abcam user community

Verified customer

Submitted Mar 10 2017

Question

1.Can any of the components be stored when diluted or must they be used immediately?
2. At what temperature should diluted components be stored?
3. If the detection antibody is conjugated, why should it be kept frozen?

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Answer

Thank you for contacting us.
Here are the answers to your questions:
1. After dilution, each diluted component should be used within the same day.
2. At 4C for same day.
3. for longer time storage, as it will not be frozen at -20C.
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

1. How many cells or mg of tissue will yield 4-20ug of nuclear extract?
2. Can customer perform this assay using their own 96-well plate? Maybe after avidin coating?
3. The protocol says to read the absorbance within 2-15 minutes? Would it be detrimental to read the signal after 15 minutes? Does the signal fade or for how much longer is it stable?
4. If a plate reader does not read at 450nm, but at 470nm, will that still work?
5. If a researcher is concerned about low detection: Can an overnight incubation of the nuclear extracts with assay buffer (step 3) be done? Or could another step be done overnight, e.g. the capture antibody incubation?

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Answer

Thank you for contacting us.
Here are the answers to your questions:
1. 100,000-500,000 cells or 10-50 mg tissues.
2. It is possible but results are not guaranteed.
3. It can be stable for 30 min after adding stop solution.
4. It may not work at 470 nm.
5. Enzyme or nuclear extract reaction step should be finished within 1-3 h at 37C. Capture antibody step could be overnight at 4C. However the background may be increased.
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

wants to use rat oligodendrocytes with and without inhibitors for MT
1) is the kit suitable for these conditions?
2) image on datasheet: was done specifically done for G9a. How was it specifically measured for this methyltransferase while excluding the other methyltransferases?
3) testing discount possible?
4) antibodies are specific to which target?

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Answer

Thank you for contacting us and thank you for your patience.

I have heard back from the lab with the following information:

1. Yes, this kit is suitable for the conditions mentioned (rat oligodendrocytes with and without inhibitors for MT) if the sufficient amount of nuclear protein can be obtained from rat oligodendrocytes andis used for each assay point ( 3-5 ug). Rat samples have however not yet been tested, but should work theoretically.

2. Purified G9a was spiked into the nuclear extract that has low concentration of nuclear proteins by dilution - for obtaining the image.

3. Unfortunately, a testing discount is not possible at the moment for this kind of kits. We offer them for antibodies and proteins as well as ELISA kits. I'm sorry about this.

4. The antibodies are specific to methylated H3 at lysine site 9.

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

I am interested in using this kit to study methyltransferase activity in a species of fly. Do you know if anyone has successfully used it with Drosophila? Thanks.

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Answer

Thanks for your inquiry. Yes, the kit ab113453 is suitable for measuring H3K9 activity from various species including Drosophila based on the working principle and antibody's specificity. Please let me know if you require further assistance.

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