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    products/chip-kits/histone-acetyltransferase-activity-assay-kit-colorimetric-ab65352.pdf

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Epigenetics and Nuclear Signaling Chromatin Modifying Enzymes Acetylation HAT
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Histone Acetyltransferase Activity Assay Kit (Colorimetric) (ab65352)

  • Datasheet
  • SDS
  • Protocol Booklet
Reviews (1)Q&A (8)References (42)

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Functional studies - ab65352
  • Functional studies - ab65352
  • Functional Studies - Histone Acetyltransferase Activity Assay Kit (Colorimetric) (ab65352)

Key features and details

  • Assay type: Enzyme activity
  • Detection method: Colorimetric
  • Platform: Microplate reader
  • Sample type: Nuclear Extracts, Purified protein, Tissue Lysate

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Overview

  • Product name

    Histone Acetyltransferase Activity Assay Kit (Colorimetric)
    See all Histone acetyltransferase kits
  • Detection method

    Colorimetric
  • Sample type

    Nuclear Extracts, Purified protein, Tissue Lysate
  • Assay type

    Enzyme activity
  • Species reactivity

    Reacts with: Mammals, Other species
  • Product overview

    Abcam's Histone Acetyltransferase Activity Assay Kit offers a convenient, nonradioactive system for a rapid and sensitive detection of HAT activity in mammalian samples. The kit utilizes active Nuclear Extract (NE) as a positive control and acetyl-CoA as a cofactor. Acetylation of peptide substrate by active HAT releases the free form of CoA which then serves as an essential coenzyme for producing NADH. NADH can easily be detected spectrophotometrically upon reacting with a soluble tetrazolium dye. The detection can be continuous and suitable for kinetic studies. The kit provides a simple, straightforward protocol for a complete assay.
    Visit our FAQs page for tips and troubleshooting.


    HAT assay protocol summary:
    - add samples to wells
    - add HAT assay mix
    - incubate for 1-4 hrs
    - analyze with microplate reader

  • Notes

    This product is manufactured by BioVision, an Abcam company and was previously called K332 HAT Activity Colorimetric Assay Kit. K332-100 is the same size as the 100 test size of ab65352.

    Histone acetyltransferases (HATs) have been implicated to play a crucial role in various cellular functions, such as gene transcription, differentiation, and proliferation.

    Abcam has not and does not intend to apply for the REACH Authorisation of customers’ uses of products that contain European Authorisation list (Annex XIV) substances.
    It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Store at -80°C. Please refer to protocols.
  • Components Identifier 100 tests
    2X HAT Assay Buffer Amber 1 x 7.5ml
    HAT Reconstitution Buffer Clear 1 x 1.8ml
    HAT Substrate I Blue 1 vial
    HAT Substrate II Red 1 vial
    NADH Generating Enzyme Green 1 x 800µl
    Nuclear Extract (4 mg/ml ) Violet 1 x 50µl
  • Research areas

    • Epigenetics and Nuclear Signaling
    • Chromatin Modifying Enzymes
    • Acetylation
    • HAT
    • Kits/ Lysates/ Other
    • Kits
    • Cell Metabolism Kits
    • Amino Acid Assays
    • Kits/ Lysates/ Other
    • Kits
    • Epigenetic kits
    • Histone acetylation and deacetylation
    • Epigenetics and Nuclear Signaling
    • Assays & Kits
    • Histone acetylation and deacetylation
  • Relevance

    Histone acetyltransferases (HAT) are enzymes that acetylate conserved lysine amino acids on histone proteins. They have been implicated to play a crucial role in various cellular functions, such as gene transcription, differentiation, and proliferation.
  • Cellular localization

    Cytoplasmic and Nuclear. Note=Nuclear in S-phase cells and cytoplasmic.
  • Alternative names

    • HAT
    • HAT1
    • Histone acetyltransferase 1
    • Histone acetyltransferase type B catalytic subunit
    • KAT1
    see all

Associated products

  • Related Products

    • Nuclear Extraction Kit (ab113474)

Images

  • Functional studies - ab65352
    Functional studies - ab65352Image from Goriki A et al., PloS Biol 12(4), Figure S11. doi:10.1371/journal.pbio.1001839. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

    HAT activity in NIH3T3 cells, which were transfected with the desired plasmids by using Lipofectamine 2000. After 24 hours from transfection, the nuclear protein was extracted and the Histone acetyltransferase activity was measured by using ab65352. 

  • Functional studies - ab65352
    Functional studies - ab65352Image from Jha et al., PLoS One 12(5), fig 2C. doi: 10.1371/journal.pone.0177372. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

    Histone acetylation activity assay for L.donovani cells over-expressing HAT1, HAT2, HAT3 and HAT4. The activities were compared to wild-type (WT) cells and pLPneo2 (vector) only transfected cells. The relative activity was determined using Histone Acetyltransferase acitvity assay kit (ab65352). 

  • Functional Studies - Histone Acetyltransferase Activity Assay Kit (Colorimetric) (ab65352)
    Functional Studies - Histone Acetyltransferase Activity Assay Kit (Colorimetric) (ab65352)
    Analysis of Histone Acetyltransferase Activity in HeLa Nuclear Extract using ab65352. HeLa nuclear extract in various amounts was incubated with HAT substrate. Activity was analyzed in a micro plate reader at 440 nm according to the kit instructions.

Protocols

  • Protocol Booklet

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (42)

Publishing research using ab65352? Please let us know so that we can cite the reference in this datasheet.

ab65352 has been referenced in 42 publications.

  • Rocha MA  et al. RNAP II produces capped 18S and 25S ribosomal RNAs resistant to 5'-monophosphate dependent processive 5' to 3' exonuclease in polymerase switched Saccharomyces cerevisiae. BMC Mol Cell Biol 23:17 (2022). PubMed: 35399070
  • Waghmare SG  et al. Histone Acetylation Dynamics during In Vivo and In Vitro Oocyte Aging in Common Carp Cyprinus carpio. Int J Mol Sci 22:N/A (2021). PubMed: 34204879
  • Lim Y  et al. P300/CBP-Associated Factor Activates Cardiac Fibroblasts by SMAD2 Acetylation. Int J Mol Sci 22:N/A (2021). PubMed: 34576109
  • Davegårdh C  et al. VPS39-deficiency observed in type 2 diabetes impairs muscle stem cell differentiation via altered autophagy and epigenetics. Nat Commun 12:2431 (2021). PubMed: 33893273
  • Chen Q  et al. Acetyl-CoA derived from hepatic mitochondrial fatty acid ß-oxidation aggravates inflammation by enhancing p65 acetylation. iScience 24:103244 (2021). PubMed: 34746707
View all Publications for this product

Customer reviews and Q&As

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1-9 of 9 Abreviews or Q&A

Rat liver nuclear fraction

Excellent Excellent 5/5 (Ease of Use)
Abreviews
Abreviews
Our samples were not within the linear range for the booklet's recommended protein concentration. A preliminary serial dilution curve should be prepared prior to running the full assay so as to ensure OD values are within the linear range.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Apr 01 2015

Question

how to remove coenzyme A and NADH?

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Abcam community

Verified customer

Asked on Jun 22 2012

Answer

You can use a background control sample, also use a nuclear extract.

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Abcam Scientific Support

Answered on Jun 22 2012

Question

Customer kindly called to discuss whether they might use a purificed sample in lieu of nuclear sample. Specifically they wish to perform an IP with their sample elute the protein of interest using a FLAG tag and then running the Histone Acetyltransferase  Activity Assay. Although they are aware this kit has not been tested in these conditions, they wish to know if this sample type might work and if by using this sample type they might have to adjust other parts of the protocol.

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Abcam community

Verified customer

Asked on May 21 2012

Answer

I have had the chance to inquire to the lab about the use of a purified IP sample. Theywould suggest using the initial sample without any IP for the HAT assay. If the IP is just for concentrating the sample, they recommend use of spin columns, not IP.

Theoretically though, the IP samples should also work they do not have any experience using this method and though modifications to the protocol may have to be performed to optimize the reaction, these would have to be determine experimentally in the lab.

Read More

Abcam Scientific Support

Answered on May 21 2012

Question

Can this Histone Acetyltransferase Activity Assay Kit (Colorimetric) also be used to measure Tubulin Acetyltransferase activity?

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Abcam community

Verified customer

Asked on Dec 19 2011

Answer

The lab has confirmed that this kit has not been validated for measuring Tubulin Acetyltransferase activity.  We have no information about whether it would be suitable for this purpose, but the kit was specifically designed for measuring Histone Acetyltransferase Activity. 

Read More

Abcam Scientific Support

Answered on Dec 19 2011

Question

1. how to calculate the OD and results?   2. the source of "1x50ul Nuclear Extract(4mg/ml)". Is this HeLa cell extract?   3. Is it possible to purchase Nuclear Extract component of this kit?  

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Abcam community

Verified customer

Asked on Aug 16 2011

Answer

Thank you very much for your inquiry. 1.) I can confirm that the HAT activity can be expressed as the relative O.D. value per µg or nmol/min/µg sample. Please have a look at the following example calculation: A. Relative OD/ug: One obtains two readings at two time points in a linear region (from measuring OD kinetically) Example: suppose background (BG) and Sample (S) gave readings of 0.1 and 0.25 at T = 3 min and 0.12 and 0.6 at T = 30 min and sample was at 50 ug in 40 ul water. Then OD2 = 0.48 and OD1 = 0.15 (after subtracting background) so Rel Activity = (0.48-0.15)/(27*50) = 0.33 per min per ug. (27 is the time interval 30 min - 3 min) B. For nmol/min/ug: Need to first determine pathlength-which depends on the 96-well plate used and any meniscus from the buffers so: 1. First take any solution in the 1X HAT assay buffer (just dilute 1:1 with water) and adjust to read 1.0 OD in a 1 cm cuvette at a given wavelength (let’s say it was Coomassie blue and you measured at 595 nm); then take exactly 108 ul and place in a well of the 96-well plate you are using and measure the absorbance at 595 nm (and let’s suppose you get 0.25); so at a path length of 1 cm you got OD =1, at the well depth pathlength you got 0.25; so the pathlength is 0.25 cm 2. Now you know the pathlength in this buffer (which accounts for a meniscus effect from any detergent, etc. in the buffer). So Measure as in (a) above and let’s assume you get the same values as in (a) above: Then the diff in OD = 0.48-0.15 = 0.33; the time interval was 27 min; the amount of protein was 50 ug; the total volume was 108 ul and the datasheet tells you the extinction coefficient is 37000 M-1 cm-1. So: amount of product = (108 ul) x ((0.33)/(37000 x 0.25)) x (1L/1000000 ul) x (1000000000 nmol/mol)= 3.853 nmol and this was measured over 27 min and using 50 ug so = 3.853 nmol/(27 min x 50 ug) = 0.00285 nmol/min/ug. OR to convert to a common U (umol/min) and per mg would get the same value since 1000 nmol/umol and 1000 ug/mg cancel each other out so = 0.00285 U/mg 2.) I can confirm that the source of the NE buffer is Jurkat cells. 3.) I am sorry to confirm that we do not offer a Jurkat cell lysate suitable for this purpose at the moment. But I would like to reassure you that you will not need it for this kit, because the NE buffer acts as a positive control.

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Abcam Scientific Support

Answered on Aug 16 2011

Question

  https://www.abcam.com/Histone-Acetyltransferase-Activity-Assay-Kit-Colorimetric-ab65352.html   My sample is histone which is extracted with acidic buffer.   This acidic buffer includes DDT so, would like to ask how can it remove be removed from the histone sample.   If you have a protocol for this situation, or whole protocol for sample preparation,  please let me know.   

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Abcam community

Verified customer

Asked on Aug 12 2011

Answer

DDT should be avoided, as this compound strongly interferes with the reactions in the kit. There are a few things I would like to suggest: - After acid extracting the histones in your current protocol, the sample could be dialysed overnight in 1 x TBS with 3,500 MWCO dialysis tubing. This would neutralize the sample. - You could use our nuclear fractionation protocol, but exclude DTT from the buffers. Here is a link to the protocol: https://www.abcam.com/index.html?pageconfig=resource&rid=11408 - Our cell fractionation kit, ab109719 Cell Fractionation Kit Standard, could be used: https://www.abcam.com/Cell-Fractionation-Kit-Standard-ab109719.html The nuclear extraction buffer in this kit contains SDS and DTT to generate a nuclear fraction for WB. Therefore this fraction is not compatible with a subsequent enzymatic analysis (though the other fractions are active). However, it may be possible to analyze the nuclear fraction without adding this buffer. Simply add water to the N fraction or treat with RIPA buffer : 1X RIPA Buffer: 20 mM Tris-HCl (pH 7.5) 150 mM NaCl, 1 mM Na2EDTA 1 mM EGTA 1% NP-40 1% sodium deoxycholate 2.5 mM sodium pyrophosphate 1 mM b-glycerophosphate 1 mM Na3VO4 1 μg/ml leupeptin

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Abcam Scientific Support

Answered on Aug 12 2011

Question

Can we use the tissue which we have frozen in liquid nitrogen? They are whole lung tissue and not nuclear fractions. Is it possible to use the kit on whole cell extracts?

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Abcam community

Verified customer

Asked on Apr 01 2011

Answer

As long as the tissue samples were frozen appropriately, nuclear lysates generated from these tissues can be used for the HAT assay.

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Abcam Scientific Support

Answered on Apr 01 2011

Question

Using lysis buffer which contains b-mercaptoethanol. Would this have an effect on the results?

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Abcam community

Verified customer

Asked on Mar 15 2011

Answer

The feedback from the lab is that technically BME should be fine but we have not tested this specifically.

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Abcam Scientific Support

Answered on Mar 15 2011

Question

I have some questions about this product. 1. How many samples can I run for each kit? (if duplicate samples) 2. Our lab has only spectrometer at 405nm. Is this OK to read the result? 3. Can I use whole cell abstract instead of nuclear extract?

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Abcam community

Verified customer

Asked on Feb 15 2010

Answer

Thank you for your enquiry. I have obtained the following information to help answer your questions: 1) Each kit can test 50 samples. i.e. 100/2, minus the wells for background and the Hela nuclear extract used for a standard 2) If you use a spectrophotometer at 405 nm, whether you will be able to accurately read the results will depend on the bandwidth of the filter your reader has. If it has a 30 nm bandwidth that would cover 440 nm; if the band width is very small, may not be optimal. I can recommend to check this. 3) I can suggest that whole cell extracts will probably give more background and the results may not be as clear. As HAT is a nuclear protein, we would recommend to try nuclear extract.

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Abcam Scientific Support

Answered on Feb 15 2010

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