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Epigenetics and Nuclear Signaling Chromatin Modifying Enzymes Acetylation
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SIRT3 Activity Assay Kit (Fluorometric) (ab156067)

  • Datasheet
  • SDS
  • Protocol Booklet
Reviews (1)Q&A (3)References (8)

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ab156067 - SIRT3 Activity Assay Kit (Fluorometric)
  • ab156067 - SIRT3 Activity Assay Kit (Fluorometric)

Key features and details

  • Assay type: Enzyme activity
  • Detection method: Fluorescent
  • Platform: Microplate reader
  • Assay time: 40 min
  • Sample type: Cell culture extracts, Tissue Extracts

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Overview

  • Product name

    SIRT3 Activity Assay Kit (Fluorometric)
  • Detection method

    Fluorescent
  • Sample type

    Cell culture extracts, Tissue Extracts
  • Assay type

    Enzyme activity
  • Assay time

    0h 40m
  • Species reactivity

    Reacts with: Human
  • Product overview

    Abcam’s SIRT3 Activity Assay Kit (Fluorometric) (ab156067) detects SIRT3 activity in lysates.


    Primarily, the SIRT3 Activity Assay Kit (Fluorometric) is designed for the rapid and sensitive evaluation of SIRT3 inhibitors or activators using crude SIRT3 fraction or purified SIRT3.


    Applications for this kit include:


    1. Screening inhibitors or activators of SIRT3.


    2. Detecting the effects of pharmacological agents on SIRT3.


    SIRT3 assay protocol summary:
    - add assay buffer, substrate peptide and NAD to wells
    - add developer to wells
    - add enzyme sample or recombinant SIRT3 to wells
    - analyze with microplate reader for 30-60 min every 1-2 min

  • Notes

    Histone Deacetylases (HDACs) are a class of enzymes responsible for the deacetylation of lysine residues on the N-terminal part of the core histones (H2A, H2B, H3 and H4), allowing the histones to wrap the DNA more tightly.

    HDAC proteins occur in four groups (class I, class IIA, class IIB, class III, class IV) based on function and DNA sequence similarity.
    Classes I, IIA and IIB are considered "classical" HDACs whose activities are inhibited by trichostatin A (TSA), whereas class III is a family of NAD+-dependent proteins (sirtuins) not affected by TSA. Class IV is considered an atypical class on its own, based solely on DNA sequence similarity to the others.

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Please refer to protocols.
  • Components 100 tests
    Developer 1 x 500µl
    Fluoro-Deacetylated Peptide (0.2 mM) 1 x 100µl
    Fluoro-Substrate Peptide (0.2 mM) 1 x 500µl
    NAD (2 mM) 1 x 500µl
    Recombinant SIRT3 1 x 500µl
    SIRT Assay Buffer 2 x 1ml
    Stop Solution 2 x 1ml
  • Research areas

    • Epigenetics and Nuclear Signaling
    • Chromatin Modifying Enzymes
    • Acetylation
    • Cell Biology
    • Cell Cycle
    • Cell Division
    • Other cell division antibodies
    • Epigenetics and Nuclear Signaling
    • Chromatin Modifying Enzymes
    • Acetylation
    • HDACs
    • Class III / Sir2 class
    • Cancer
    • Oncoproteins/suppressors
    • Tumor suppressors
    • Other
    • Cardiovascular
    • Heart
    • Cardiac metabolism
    • Epigenetics and Nuclear Signaling
    • Chromatin Modifying Enzymes
    • Acetylation
    • HDACs
    • Kits
    • Kits/ Lysates/ Other
    • Kits
    • Cell Metabolism Kits
    • Other Metabolism Assay
    • Kits/ Lysates/ Other
    • Kits
    • Epigenetic kits
    • Histone acetylation and deacetylation
    • Metabolism
    • Types of disease
    • Obesity
  • Function

    NAD-dependent protein deacetylase. Activates or deactivates mitochondrial target proteins by deacetylating key lysine residues. Known targets include ACSS1, IDH, GDH, SOD2, PDHA1, LCAD, SDHA and the ATP synthase subunit ATP5O. Contributes to the regulation of the cellular energy metabolism. Important for regulating tissue-specific ATP levels.
  • Tissue specificity

    Widely expressed.
  • Sequence similarities

    Belongs to the sirtuin family. Class I subfamily.
    Contains 1 deacetylase sirtuin-type domain.
  • Post-translational
    modifications

    Processed by mitochondrial processing peptidase (MPP) to give a 28 kDa product. Such processing is probably essential for its enzymatic activity.
  • Cellular localization

    Mitochondrion matrix.
  • Target information above from: UniProt accession Q9NTG7 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Alternative names

    • hSIRT 3
    • hSIRT3
    • Mitochondrial nicotinamide adenine dinucleotide dependent deacetylase
    • NAD dependent deacetylase sirtuin 3 mitochondrial
    • NAD-dependent protein deacetylase sirtuin-3, mitochondrial
    • Regulatory protein SIR2 homolog 3
    • Silent mating type information regulation 2 S.cerevisiae homolog 3
    • Sir 2 like 3
    • SIR 2 like protein 3
    • SIR 3
    • SIR2 L3
    • Sir2 like 3
    • SIR2 like protein 3
    • SIR2-like protein 3
    • SIR2L3
    • SIR3_HUMAN
    • SIRT 3
    • SIRT3
    • Sirtuin 3
    • Sirtuin silent mating type information regulation 2 homolog 3 (S. cerevisiae)
    • Sirtuin type 3
    • Sirtuin3
    see all
  • Database links

    • Entrez Gene: 23410 Human
    • Omim: 604481 Human
    • SwissProt: Q9NTG7 Human
    • Unigene: 716456 Human

    Images

    • ab156067 - SIRT3 Activity Assay Kit (Fluorometric)
      ab156067 - SIRT3 Activity Assay Kit (Fluorometric)
      Dose dependency curve of recombinant SIRT3 activity
    • ab156067 - SIRT3 Activity Assay Kit (Fluorometric)
      ab156067 - SIRT3 Activity Assay Kit (Fluorometric)
      Effect of Trichostatin A and NAD on recombinant SIRT3 activity

    Protocols

    • Protocol Booklet

    Click here to view the general protocols

    Datasheets and documents

    • SDS download

    • Datasheet download

      Download

    References (8)

    Publishing research using ab156067? Please let us know so that we can cite the reference in this datasheet.

    ab156067 has been referenced in 8 publications.

    • Botezelli JD  et al. Adipose depot-specific upregulation of Ucp1 or mitochondrial oxidative complex proteins are early consequences of genetic insulin reduction in mice. Am J Physiol Endocrinol Metab 319:E529-E539 (2020). PubMed: 32715748
    • Ouyang J  et al. SIRT3 Inactivation Promotes Acute Kidney Injury Through Elevated Acetylation of SOD2 and p53. J Surg Res 233:221-230 (2019). PubMed: 30502252
    • Miao HH  et al. Ginsenoside Rg1 attenuates isoflurane/surgery-induced cognitive disorders and sirtuin 3 dysfunction. Biosci Rep 39:N/A (2019). PubMed: 31652451
    • Yi X  et al. SIRT3-Dependent Mitochondrial Dynamics Remodeling Contributes to Oxidative Stress-Induced Melanocyte Degeneration in Vitiligo. Theranostics 9:1614-1633 (2019). PubMed: 31037127
    • Chen LY  et al. Activation of AMPK-SIRT3 signaling is chondroprotective by preserving mitochondrial DNA integrity and function. Osteoarthritis Cartilage 26:1539-1550 (2018). PubMed: 30031925
    • Yin J  et al. Sirtuin 3 attenuates amyloid-ß induced neuronal hypometabolism. Aging (Albany NY) 10:2874-2883 (2018). PubMed: 30362958
    • Michalak S  et al. Mitochondrial Respiration in Intact Peripheral Blood Mononuclear Cells and Sirtuin 3 Activity in Patients with Movement Disorders. Oxid Med Cell Longev 2017:9703574 (2017). PubMed: 29081897
    • Kwon S  et al. Obesity and aging diminish sirtuin 1 (SIRT1)-mediated deacetylation of SIRT3, leading to hyperacetylation and decreased activity and stability of SIRT3. J Biol Chem 292:17312-17323 (2017). PubMed: 28808064

    Customer reviews and Q&As

    Show All Reviews Q&A
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    1-4 of 4 Abreviews or Q&A

    Sirt3 activity in liver of Wistar rart

    Good Good 4/5 (Ease of Use)
    Abreviews
    Abreviews
    abreview image
    While Sirt 3 activity was easy to measure with the recombinant enzyme, RFU levels from crude liver lysate of Wistar rats were very low. Finally, after several settings (buffer, fractionation, washing steps, etc...), sirt 3 activity was detected in liver of Wistar rats, under our specific experimental conditions.
    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.

    MS. Karine Bardin

    Verified customer

    Submitted Jul 06 2016

    Question

    I would like to use this kit (ab156067 sirt3 assay kit), in order to measure endogenous SIRT3 activity in cell. According to the cover sleeve the kit is intended " For the quantitative measurement of SIRT3 activity in cell lysates".
    However, after reading the booklet, i understand that is not possible to measure endogenous cellular SIRT3 activity with this kit, which only detects activity of recombinant or immunoprecipitated SIRT3 protein.
    Is that right? Can you confirm my question?

    Read More

    Abcam community

    Verified customer

    Asked on Sep 15 2015

    Answer



    I am happy to confirm that crude cell lysate can be used as samples with this kit but as this often needs a lot of optimisation we would recommend to prepare mitochondria and use a mitochondrial lysate.

    I can recommend to use one of our mitochondria isolation kits for this purpose:

    ab110171 (Mitochondria Isolation Kit for Cultured Cells (with Dounce Homogenizer))

    https://www.abcam.com/Mitochondria-Isolation-Kit-for-Cultured-Cells-with-Dounce-Homogenizer-ab110171.html (or use the following: https://www.abcam.com/Mitochondria-Isolation-Kit-for-Cultured-Cells-with-Dounce-Homogenizer-ab110171.html).

    ab110169 (Mitochondria Isolation Kit for Tissue (with Dounce Homogenizer))

    https://www.abcam.com/Mitochondria-Isolation-Kit-for-Tissue-ab110168.html (or use the following: https://www.abcam.com/Mitochondria-Isolation-Kit-for-Tissue-with-Dounce-Homogenizer-ab110169.html).

    Read More

    Anja Hoffmann

    Abcam Scientific Support

    Answered on Sep 15 2015

    Question


    1) How specific is the assay? There are two other SIRT proteins in the mitochondria, and both are NAD dependent. It seems as if this assay will measure activity of all of these proteins, no?
    2) What is the difference between the mitochondrial isolation kit and using a normal Triton/Digitonin protocol for mitochondrial extraction?
    3) Are intact mitochondria required for the SIRT3 activity assay, or are mitochondrial extracts enough?
    4) Would it be possible to receive a test sample of either of these kits, to see if they work for what I want to do before buying the whole kit?

    Read More

    Abcam community

    Verified customer

    Asked on Nov 26 2014

    Answer

    1) How specific is the assay? There are two other SIRT proteins in the mitochondria, and both are NAD dependent. It seems as if this assay will measure activity of all of these proteins, no?
    I assume you are referring to SIRT5 as the second SIRT protein? We know that SIRT1 (recombinant) can catalyze deacetylation from the substrate of SIRT3 Activity Assay Kit (Fluorometric) and SIRT2 cannot. We don't know about SIRT5.

    2) What is the difference between the mitochondrial isolation kit and using a normal Triton/Digitonin protocol for mitochondrial extraction?
    The kit comes with optimized detergent concentrations to isolate mitochondria and an optimized isosmotic buffer to reduce swelling.

    3) Are intact mitochondria required for the SIRT3 activity assay, or are mitochondrial extracts enough?
    We haven't used SIRT3 Activity Assay Kit (Fluorometric) with native SIRT3 samples ourselves.

    4) Would it be possible to receive a test sample of either of these kits, to see if they work for what I want to do before buying the whole kit?
    Unfortunately, we do not offer test samples of our products due to logistical issues. However, all our products are covered by our 1 year Abpromise guarantee which states that if the product does not work as stated on the datasheet, we will replace or refund the product.

    Read More

    Elisa Thomas

    Abcam Scientific Support

    Answered on Nov 26 2014

    Question

    Is the Recombinant SIRT3 made from baculovirus? Is it full lenght or fragment?

    Read More

    Abcam community

    Verified customer

    Asked on Nov 21 2013

    Answer

    The recombinant SIRT3 was made/refined from colon bacillus. It is a fragmented protein from the N terminal, plus made to be around 10kDa size (process was not carried out within the cell).

    Read More

    達偉 鄭

    Abcam Scientific Support

    Answered on Nov 21 2013

    Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
    For licensing inquiries, please contact partnerships@abcam.com

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