SIRT3 Activity Assay Kit (Fluorometric) (ab156067)

Reviews (1)Q&A (3)References (2)

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

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

References

This product has been referenced in:
  • Chen LY  et al. Activation of AMPK-SIRT3 signaling is chondroprotective by preserving mitochondrial DNA integrity and function. Osteoarthritis Cartilage 26:1539-1550 (2018). Read more (PubMed: 30031925) »
  • 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). Read more (PubMed: 29081897) »
See all 2 Publications for this product

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

Customer reviews and Q&As

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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
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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.

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?

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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/index.html?datasheet=110171 (or use the following: https://www.abcam.com/index.html?datasheet=110171).

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

https://www.abcam.com/index.html?datasheet=110168 (or use the following: https://www.abcam.com/index.html?datasheet=110169).

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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?

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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.

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Question

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

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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).

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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