SIRT1 Activity Assay Kit (Fluorometric) (ab156065)

Reviews (1)Q&A (4)References (21)

Overview

  • Product name
    SIRT1 Activity Assay Kit (Fluorometric)
    See all SIRT1 kits
  • Detection method
    Fluorescent
  • Sample type
    Cell culture extracts, Tissue Extracts
  • Assay type
    Enzyme activity
  • Assay time
    1h 00m
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Product overview

    SIRT1 Activity Assay Kit (Fluorometric) (ab156065) detects SIRT1 activity in lysates. Primarily, the SIRT1 Activity Assay Kit is designed for the rapid and sensitive evaluation of SIRT1 inhibitors or activators using crude SIRT1 fraction or purified SIRT1. Additionally, any cultured primary cell, cell line, or tissue homogenate can be assayed for SIRT1 activity with the SIRT1 Activity Assay Kit if the appropriate antibody directed against SIRT1 (Anti-SIRT1 antibody (ab7343)) is used for immunoprecipitation.

    Abcam’s SIRT1 Activity Assay Kit (Fluorometric) has been shown to detect the activity of Sirtuins, at least SIRT1 in Human or animal cell lysates or in column fractions. The assay shows good linearity of sample response. The assay may be used to follow the purification of Sirtuins or may be used to detect the presence of Sirtuins in cell lysates.

    Applications for this kit include:

    1. Monitoring the purification of SIRT1.

    2. Screening inhibitors or activators of SIRT1.

    3. Detecting the effects of pharmacological agents on SIRT1.

  • 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.

     

Properties

Associated products

Images

  • SIRT1 Activity Assay Kit (ab156065)
    SIRT1 Activity Assay Kit (ab156065)

    Time course of SIRT1-substrate deacetylation by recombinant SIRT1 in the presence of EX-527 (ab141506)

  • SIRT1 Activity Assay Kit (ab156065)
    SIRT1 Activity Assay Kit (ab156065)

    Time course of SIRT1-substrate deacetylation by recombinant SIRT1

  • ab156065 - SIRT1 Activity Assay Kit (Fluorometric)
    ab156065 - SIRT1 Activity Assay Kit (Fluorometric)
    Dose dependency curve of recombinant SIRT1 activity
  • ab156065 - SIRT1 Activity Assay Kit (Fluorometric)
    ab156065 - SIRT1 Activity Assay Kit (Fluorometric)

    Measurement of 293T cell endogenous SIRT1 activity in a sample previously immunoprecipitated with an anti-SIRT1 antibody.

Protocols

References

This product has been referenced in:
  • Ma CH  et al. Homocysteine causes dysfunction of chondrocytes and oxidative stress through repression of SIRT1/AMPK pathway: A possible link between hyperhomocysteinemia and osteoarthritis. Redox Biol 15:504-512 (2018). Read more (PubMed: 29413962) »
  • Yao ZQ  et al. A novel small-molecule activator of Sirtuin-1 induces autophagic cell death/mitophagy as a potential therapeutic strategy in glioblastoma. Cell Death Dis 9:767 (2018). Read more (PubMed: 29991742) »
See all 21 Publications for this product

Publishing research using ab156065? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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1-5 of 5 Abreviews or Q&A

Use the ab156065: SIRT1 activity assay kit for human serum samples.

 Good Good 4/5 (Ease of Use)
Abreviews
abreview image
We use fresh and frozen human serum (n=9).
Blood was collected in purple tubes (with EDTA) and centrifuged for 1500 rpm x 15 minutes for separation.
Part of serum was frozen for 45 minutes, other part kept on ice.
For test we used 30uL of serum. We found no difference in RFU of both samples.
Volumes of reagent are small, it is hard to load in black plate.
It will be nice to have calibration curve for sirt1 activity.

Abcam user community

Verified customer

Submitted Jan 25 2016

Question

In the user protocol, page 11, 8. Assay protocol step 2: "Add Developer (#5) to each well of the
microtiter plate and mix well" Have you tried that if the steps 1 and 2 would be combined? I mean that if I would add developer
already in the Step 1 solution? Because now, if I would follow the assay protocol, there would be a significant time delay in hand pipeting.

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Answer



You can of course add Distilled water, SIRT1 Assay Buffer, Fluoro-Substrate Peptide, NAD and the Developer at the same time rather than the developer after.

Please make sure when doing this, that all volumes of distilled water added is the same in all of your assays.

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Question

I want to know about the role of EDTA in sample preparation? I read in the protocol text that EDTA has an inhibitory effect on divalent cation-dependent proteases and it also says that we shouldn't use protease inhibitors.

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Answer

Many biological and biochemical buffers contain a small amount of EDTA as it has many effects via chelating. A large amount of EDTA will completely inhibit the activities of some enzymes, but a small amount of EDTA helps prevent oxidation on proteins. The small amount of EDTA in the extraction buffer doesn't have an effect on the activity of the protease used in the kit, but it does help to prevent oxidation.

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Question

Can SIRT1 activity be distinguished without immunoprecipitation of SIRT1?

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Answer



Please note there is no way to distinguish SIRT1 activity from other SIRT family members (mainly SIRT3) without immunoprecipitation strictly. Having said that, SIRT1 is distributed in the cell nucleus and SIRT3 is in the mitochondria. So in theory you can use subcellular fractionation of nucleus and mitochondria to separate the activities. However this method would not be very precise as there are other family members whose activity need to be taken into account and subcellular fractionation is not 100% precise.

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Question

1. Is Trichostatin A provided with this kit?

2. Is immunoprecipitation required to measure SIRT1 activity?

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Answer

1. In manual of this kit ,it has been mentioned “this kit is designed so
that the activity of NAD dependent Histone deacetylase can be measured
under existence of Trichostatin A, which is the powerful inhibitor of
HDACs”,but trichostatin A has not been supplied in this kit.


Trichostatin A isn't required for this kit, because it is already added to some of kit components.

2. I would like to know If trichostatin A is necessary in order to measurement
of Sirt1 in cell lysate without immunopercipitation?


Please note that the substrate of SIRT1 assay kit is deacetylated not only by SIRT1 but also by SIRT3, and maybe others. I recommend doing immunopercipitation to meaure SIRT1-specific activity.

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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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