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566329 SIRT2 Activity Assay Kit

SIRT2 Activity Assay Kit MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
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Overview

Replacement Information

Key Spec Table

Detection Methods
Fluorometric
Description
Overview

This product has been discontinued.





The SIRT2 Activity Assay Kit is designed to screen potential inhibitors SIRT2.
Catalogue Number566329
Brand Family Calbiochem®
Application Data
Materials Required but Not Delivered• Fluorimeter for measuring fluorescence in a 96-well plate at an excitation wavelength of 350-380 nm and an emission wavelength of 440-460 nm
• Pipetman or multi-channel pipetman capable of pipetting 2-100 µl accurately
• Ice bucket to keep reagents cold until use
• Plate warmer or other temperature control device
References
ReferencesZhao, K., et al. 2004. J. Mol. Biol. 337, 731.
Dryden, S.C., et al. 2003. Mol. Cell Biol. 23, 3173.
North, B.J., et al. 2003. Mol. Cell 11, 437.
Zhao, K., et al. 2003. Nat. Struct. Biol. 10, 864.
Product Information
Detection methodFluorometric
Form96 Tests
Format96-well plate
Kit containsSIRT2 Enzyme, SIRT2 Deacetylase Substrate, SIRT Developer Concentrate (5X), NAD+ (Sirtuin Substrate), Nicotinamide (Sirtuin Inhibitor), Suramin (Sirtuin Inhibitor), Deacetylated Standard, Sirtuin Assay Buffer, 1/2 Volume Microplate, 1/2 Volume White Microplate, and a user protocol.
Applications
Biological Information
Sample Typeinhibitors of SIRT2
Physicochemical Information
Dimensions
Materials Information
Toxicological Information
Safety Information according to GHS
Safety Information
R PhraseR: 36/38

Irritating to eyes and skin.
S PhraseS: 22-24/25-26-36-45

Do not breathe dust.
Avoid contact with skin and eyes.
In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
Wear suitable protective clothing.
In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
Product Usage Statements
Intended useThe Calbiochem® SIRT2 Activity Assay Kit is a complete assay system designed to measure the lysyl deacetylase activity of the recombinant human SIRT2 included in the kit for the purpose of inhibitor screening.
Storage and Shipping Information
Ship Code Dry Ice Only
Toxicity Multiple Toxicity Values, refer to MSDS
Storage ≤ -70°C
Storage ConditionsUpon arrival store the 1/2 Volume Microplates at room temperature and the remaining components at -70°C.
Avoid freeze/thaw Avoid freeze/thaw
Do not freeze Ok to freeze
Packaging Information
Transport Information
Supplemental Information
Kit containsSIRT2 Enzyme, SIRT2 Deacetylase Substrate, SIRT Developer Concentrate (5X), NAD+ (Sirtuin Substrate), Nicotinamide (Sirtuin Inhibitor), Suramin (Sirtuin Inhibitor), Deacetylated Standard, Sirtuin Assay Buffer, 1/2 Volume Microplate, 1/2 Volume White Microplate, and a user protocol.
Specifications
Global Trade Item Number
Catalogue Number GTIN
566329 0

Documentation

SIRT2 Activity Assay Kit Certificates of Analysis

TitleLot Number
566329

References

Reference overview
Zhao, K., et al. 2004. J. Mol. Biol. 337, 731.
Dryden, S.C., et al. 2003. Mol. Cell Biol. 23, 3173.
North, B.J., et al. 2003. Mol. Cell 11, 437.
Zhao, K., et al. 2003. Nat. Struct. Biol. 10, 864.
User Protocol

Revision30-March-2009 JSW
Form96 Tests
Format96-well plate
Detection methodFluorometric
StorageUpon arrival store the 1/2 Volume Microplates at room temperature and the remaining components at -70°C.
Intended useThe Calbiochem® SIRT2 Activity Assay Kit is a complete assay system designed to measure the lysyl deacetylase activity of the recombinant human SIRT2 included in the kit for the purpose of inhibitor screening.
BackgroundYeast Sir2 (Silent information regulator 2) is the first-identified sirtuin, belonging to an ancient group of enzymes that is found in eukaryotes, the archaea, and eubacteria. Originally described as a factor required for maintenance of silencing at telomeres and mating-type loci, Sir2 was subsequently shown to be an enhancer of mother-cell replicative lifespan. The sirtuins represent a distinct class of trichostatin A-insensitive protein-lysyl-deacetylases (class III HDACs) and have been shown to catalyze a reaction that couples lysine deacetylation to the formation of nicotinamide and O-acetyl-ADP-ribose from NAD+ and the abstracted acetyl group. There are seven human sirtuins, which have been designated SIRT1-SIRT7. Like Sir2, human SIRT2 is a class I sirtuin, although not as closely related to Sir2 as human SIRT1. Thus far, SIRT2 is the only human sirtuin to have had its three-dimensional structure determined and its catalytic core has the same basic two-domain architecture and central groove as other sirtuins. SIRT2 is a cytoplasmic protein, although a lesser amount of nuclear localization and an interaction with the homeobox transcription factor HOXA10 have also been reported. A complex comprising HDAC6 and SIRT2 colocalizes with the microtubule network and both enzymes can deacetylate α-tubulin Lys40 in purified tubulin heterodimers or microtubules. SIRT2 protein levels rise during mitosis and it becomes phosphorylated. Overexpression of SIRT2 delays exit of cells from mitosis, suggesting that it may play a role in cell cycle regulation.
Principles of the assayThe Calbiochem® SIRT2 Activity Assay Kit is based on the unique SIRT2 Substrate/Developer combination. First, the SIRT2 Deacetylase Substrate, which comprises the p53 sequence Gln-Pro-Lys-Lys(ε-acetyl), is incubated with SIRT2 Enzyme together with the cosubstrate NAD+. Deacetylation of the SIRT2 Deacetylase Substrate sensitizes it so that, in the second step, treatment with the SIRT2 Developer produces a fluorophore. The protocols and application examples described below emphasize conditions suitable for the screening of potential inhibitors of SIRT2. Nicotinamide and Suramin are included as positive controls for SIRT2 inhibition. Although inhibitor screens are typically done at relatively low substrate concentrations, the kit does include enough substrate to perform kinetic studies over a full range of relevant concentrations. For convenience, two types of 1/2 volume 96-well plates come packaged with the kit, but the reagents have also been used successfully in other formats, including cuvettes and 384-well plates.

Figure 1: Assay Principle


Figure 2: SIRT2 Peptide Substrate Preferences

Initial rates of deacetylation were determined for a series of fluorogenic acetylated peptide substrates based on short stretches of human histone H3, H4 and p53 sequence. Recombinant human SIRT2, was incubated for 10 min at 37°C with 25 µM of the indicated fluorogenic acetylated peptide substrate and 500 µM NAD+. Reactions were stopped by the addition of Developer/2 mM nicotinamide and the deacetylation-dependent fluorescent signal was allowed to develop for 45 min. Fluorescence was then measured in the wells of a clear plate with a fluorescence plate reader (Ex. 360 nm, Em. 460 nm, gain=85).
Materials provided• SIRT2 Enzyme (Kit Component No. KP31830-500U): 1 vial, 500 U, supplied in 100 mM NaCl, 25 mM Tris-HCl, 5 mM DTT, 10% glycerol, pH 7.5; One unit is defined as the amount of enzyme required to deacetylate 1 pmol SIRT2 Deacetylase Substrate per min at 37°C (using 500 µM SIRT2 Deacetylase Substrate and 500 µM NAD+)
• SIRT2 Deacetylase Substrate (Kit Component No. KP31831-100UL): 1 vial, 100 µl, supplied as 5 mM in 137 mM NaCl, 25 mM Tris-HCl, 2.7 mM KCl, 1 mM MgCl₂, pH 8.0
• SIRT Developer Concentrate (5X) (Kit Component No. KP31832-250UL): 5 vials, 250 µl each, supplied as 5X
• NAD+ (Sirtuin Substrate) (Kit Component No. KP31833-500UL): 1 vial, 500 µl, supplied as 50 mM (oxidized form) in 137 mM NaCl, 25 mM Tris-HCl, 2.7 mM KCl, 1 mM MgCl₂, pH 8.0
• Nicotinamide (Sirtuin Inhibitor) (Kit Component No. KP31834-500UL): 1 vial, 500 µl, supplied as 50 mM in 137 mM NaCl, 25 mM Tris-HCl, 2.7 mM KCl, 1 mM MgCl₂, pH 8.0
• Suramin (Sirtuin Inhibitor) (Kit Component No. KP31835-10MG): 1 vial, 10 mg
• Deacetylated Standard (Kit Component No. KP31836-30UL): 1 vial, 30 µl, supplied as 10 mM in DMSO
• Sirtuin Assay Buffer (Kit Component No. KP31837-20ML): 1 bottle, 20 ml, supplied as 137 mM NaCl, 50 mM Tris-HCl, 2.7 mM KCl, 1 mM MgCl₂, 1 mg/ml BSA
• 1/2 Volume Microplate (Kit Component No. KP31838-1EA): 1 1/2-volume 96-well plate
• 1/2 Volume White Microplate (Kit Component No. KP31839-1EA): 1 1/2-volume white 96-well plate
Materials Required but not provided• Fluorimeter for measuring fluorescence in a 96-well plate at an excitation wavelength of 350-380 nm and an emission wavelength of 440-460 nm
• Pipetman or multi-channel pipetman capable of pipetting 2-100 µl accurately
• Ice bucket to keep reagents cold until use
• Plate warmer or other temperature control device
Precautions and recommendations• The assay is performed in two stages. The first stage, during which the SIRT2 acts on the SIRT2 Deacetylase Substrate, is done in a total volume of 50 µl. The second stage, which is initiated by the addition of 50 µl of SIRT 2 Developer, including a SIRT2 inhibitor, stops SIRT2 activity and produces the fluorescent signal.
• Two types of 1/2-volume, 96-well plates are provided with the kit. The signal obtained with the opaque, white plate can be ~5-fold greater than that obtained with the clear plate. As long as the fluorimeter to be used is configured so that excitation and emission detection occur from above the well, the white plate should significantly increase assay sensitivity.
• Should it be necessary, for convenience in adding or mixing reagents, there is some flexibility for change in the reaction volumes. The wells of the microplates provided can readily accommodate 150 µl. If planning a change to the volume of the SIRT2 Developer, it should be noted that it is important to keep two factors constant: 1) concentration of SIRT2 inhibitor (1 mM Nicotinamide) in the final mix; 2) 10 µl/well amount of SIRT2 Developer Concentrate.
• Experimental samples should be compared to a "Time Zero" (sample for which 1X SIRT2 Developer/2 mM Nicotinamide is added immediately before mixing of the SIRT2 with substrate) and/or a negative control (no enzyme).
• For many applications, including inhibitor screening, a signal approximately proportional to the initial enzyme rate is desirable. Particularly if a sub-Km substrate concentration is chosen the rate will immediately begin to decline as substrate is used up. In the case of SIRT2, inhibition by one of the reaction products, Nicotinamide, will also contribute to this effect. A preliminary time course experiment will aid in the selection of an incubation time, which yields a signal that is both sufficiently large and proportional to enzyme rate.
• The Km of SIRT2 for the SIRT2 Deacetylase Substrate has been measured at 186 µM in the presence of 3 mM NAD+. The Km for NAD+, determined in the presence of 1 mM SIRT2 Deacetylase Substrate, was 547 µM. Use of substrate concentrations at or below Km will help avoid substrate competition effects, which could mask the effectiveness of competitive inhibitors.
• The effects of some enzyme modulators, such as covalent inhibitors, may be time-dependent. In other cases, time dependence may be indicative of artifacts such as the formation of aggregates. Two schemes for order of reagent mixing are outlined in Table 1. One includes a preincubation of enzyme and test compound. The other presents substrates and test compound to the enzyme simultaneously.
• It is conceivable that some compounds being screened for modulation of SIRT2 activity may interfere with the action of the SIRT2 Developer. It is therefore important to confirm that apparent "hits" are in fact acting only via SIRT2 effects. One approach to this involves retesting the candidate compound in a reaction with the Deacetylated Standard plus the SIRT2 Developer. A detailed retesting procedure is described below. In some cases, it may be possible to avoid this retesting by means of measurements taken during the fluorescence development phase of the initial SIRT2 assay.
Reagent preparationNote: Defrost all kit components and keep these, and all dilutions described below, on ice until use. Note that it is best to rapidly thaw both the SIRT2 Enzyme and the SIRT2 Developer Concentrate (5X). All undiluted kit components are stable for several h on ice. • Diluted SIRT2 Enzyme: Assuming 5 U of SIRT2 per assay, dilute a sufficient amount to 0.2 U/µl in Assay Buffer to provide enough Diluted SIRT2 Enzyme for the number of assays to be performed (it is recommended that slightly more than the number of wells x 5 µl per well be prepared to account for pipeting error). Subsequent dilutions of five-fold to 0.2 U/µl or three fold to 0.33 U/µl will be made, depending on whether test compounds will be added with substrate or preincubated with the enzyme. • Suramin WS and Nicotinamide WS: Prepare dilution(s) of Suramin (Sirtuin Inhibitor), Nicotinamide (Sirtuin Inhibitor), and/or Test Compounds in Assay Buffer. Since 10 µl will be used per well (Table 1), and the final volume of the SIRT2 reaction is 50 µl, these inhibitor dilutions should be 5X the desired final concentrations. Suramin (Sirtuin Inhibitor) is soluble in both water and Assay Buffer (10 mg in 0.27 ml = 25 mM). High concentrations of both ethanol and DMSO will affect SIRT2 activity, therefore, appropriate solvent controls should always be included. • Substrate WS: Prepare a dilution of the substrates, SIRT2 Deacetylase Substrate and NAD+ (Sirtuin Substrate), in Assay Buffer, that is 3.33X the desired final concentrations. For inhibitor screening, substrate concentrations at or below the Km are recommended. The 3.33X stock solutions will constitute 60% of a 2X substrate stock, prepared either with or without added test compounds. • SIRT2 Developer WS: Shortly before use (<30 min.), prepare sufficient SIRT2 Developer WS containing 2 mM Nicotinamide for the number of assays to be performed; 50 µl per well is required. For example, 1 ml will contain 760 µl Assay Buffer, 200 µl SIRT2 Developer Concentrate (5X), and 40 µl 50 mM Nicotinamide (Sirtuin Inhibitor). Addition of Nicotinamide to the SIRT2 Developer insures that SIRT2 activity stops when the SIRT2 Developer WS is added. Keep SIRT2 Developer WS on ice until use.
Detailed protocolPreparation of a Standard Curve:
1. The exact concentration range of the Deacetylated Standard that will be useful for preparing a standard curve will vary depending on the fluorimeter model, the gain setting, and the exact excitation and emission wavelengths used. We recommend diluting some of the Deacetylated Standard to a relatively low concentration with Assay Buffer (1 to 5 µM). The fluorescence signal should then be determined, as described below, after mixing 50 µl of the Diluted Deacetylated Standard with 50 µl of 0.2X SIRT2 Developer. The estimate of AFU (arbitrary fluorescence units)/µM obtained with this measurement, together with the observed range of values obtained in the enzyme assays can then be used to plan an appropriate series of dilutions for a standard curve. Provided the same wavelength and gain settings are used each time, there should be no need to prepare a standard curve more than once.
2. After ascertaining an appropriate concentration range, prepare, in Assay Buffer, a series of Diluted Deacetylated Standards that span this range. Add 50 µl of each dilution and 50 µl of Assay Buffer as a 'zero', to a designated set of wells on the 1/2 Volume Microplate.
3. Prepare enough of a 0.2X SIRT2 Developer solution in Assay Buffer for addition of 50 µl to each of the Diluted Deacetylated Standard wells.
4. Add 50 µl of the 0.2X SIRT2 Developer to each of the Diluted Deacetylated Standards and incubate for 5-10 min at room temperature (25°C).
5. Read samples in a microplate-reading fluorimeter capable of excitation at a wavelength in the range 350-380 nm and detection of emitted light in the range 450-480 nm.
6. Plot fluorescence signal (y-axis) versus concentration of the Deacetylated Standard (x-axis). Determine slope as AFU/µM.

SIRT2 Activity Assay
1. Table 1 gives examples of solutions and volumes for use in various types of SIRT2 assays. These are mixtures for the first (deacetylation) phase of the assay.

Table 1: Assay Mixtures for SIRT2 Activity Assay

The Assay Buffer amount is written as a split "10 µl + 10 µl" in reference to two possibilities for the order in which test compounds are mixed with the SIRT2 enzyme: 1) If substrate and test compound are to be mixed with the enzyme simultaneously, then the entire 20 µl would be mixed with 5 µl of enzyme or a master mix consisting of 0.2 U/µl SIRT2 Enzyme in Assay Buffer could be aliquoted at 25 µl per well. In this case, substrates plus test compound (25 µl) could be added from a mother plate in which the wells contain a mixture of 40% 5X Test Compound and 60% 3.33X Substrates. 2) If the test compound is to be preincubated with enzyme prior to substrate addition, 15 µl of an enzyme master mix consisting of 0.33 U/µl SIRT2 Enzyme in Assay Buffer could be aliquoted per well and then mixed with 10 µl of 5X Test Compound. The reaction would then be initiated by addition of 25 µl Substrate WS in Assay Buffer (40% Assay Buffer, 60% 3.33X Substrates). *NOTE: In a 'Time Zero' sample, the substrate addition is made after the addition of SIRT2 Developer WS.


2. Add 25 µl of 0.2 U/µl Diluted SIRT2 Enzyme or 15 µl of 0.33 U/µl Diluted SIRT2 Enzyme and 10 µl 5X Test Compound or 25 µl Assay Buffer (solvent control) to designated individual wells of the desired 1/2 Volume Microplate.
3. Warm the Plate and Substrate WS to 37°C.
4. Initiate SIRT2 reactions by adding 25 µl Substrate WS to the wells and thoroughly mixing. DO NOT ADD SUBSTRATE TO "TIME ZERO" WELLS.
5. Allow SIRT2 reactions to proceed for desired length of time and stop the reaction by adding 50 µl SIRT2 Developer WS. Add 25 µl Substrate WS to "Time Zero" samples. Incubate plate at room temperature for at least 45 min Signal development can be accelerated by incubation at a higher temperature (30-37°C).
6. Read samples in a plate-reading fluorimeter capable of excitation at a wavelength in the range 350-380 nm and detection of emitted light in the range 450-480 nm. Completion of signal development can be assessed by taking fluorescence readings at 5 min intervals. The SIRT2 Developer reaction is complete when the fluorescence readings reach a maximum and plateau. Signals are stable for at least 60 min beyond this time.

Testing of Potential SIRT2 Inhibitors for Interference with the SIRT2 Developer or the
Fluorescence Signal:

1. The SIRT2 Developer is formulated so that, under normal circumstances, the reaction goes to completion in less than 30 min at 25°C. That, together with the recommended 45 min reaction time, should help insure that in most cases, even when some retardation of the development reaction occurs, the signal will fully develop prior to the reading of the plate.
2. A convenient step to control for substances that interfere with the SIRT2 Developer reaction or the fluorescence signal itself may be built directly into an inhibitor screening protocol. After waiting for the signal from the SIRT2 reaction to fully develop and stabilize (usually less than 45 min), the fluorescence is recorded and a 'spike' of Deacetylated Standard is added (e.g. amount equivalent to 5 µM in the 50 µl SIRT2 reaction). Sufficient SIRT2 Developer reactivity should remain to produce a full signal from this 'spike'. When the new, increased fluorescence level has fully developed (<15 min), the fluorescence is read and the difference between this reading and the first one can provide an internal standard, in terms of AFU/µM, for appropriate quantitation of each well. This is particularly useful in cases where the development reaction itself is not compromised but the fluorescence signal is diminished. Highly colored test compounds, for example, may have such an effect. As discussed further below, interference with the development reaction per se will be reflected in the kinetics of signal development, both that due to the initial SIRT2 reaction and that due to a subsequent Deacetylated Standard 'spike'.

Figure 3: Fluorescence Standard Curve

50 µl Diluted Deacetylated Standard, in Assay Buffer, at the indicated concentrations, were mixed with 50 µl 0.2X SIRT2 Developer and incubated 15 min, 25°C. Fluorescence was then measured in the wells of the clear plate with a fluorescence plate reader (Ex. 360 nm, Em. 460 nm, gain=85).


3. It should be possible to identify many cases in which there is interference with the development reaction by taking a series of fluorescence readings immediately following addition of the SIRT2 Developer (e.g. readings at 5 min intervals for 45 min). The fluorescence of control samples (no inhibitor) will change very little after the third or fourth reading. Samples containing compounds which inhibit SIRT2, but which do not interfere with SIRT2 Developer, will display similarly rapid kinetics, although a lower final fluorescence. Nicotinamide (100 µM) provides a good model of this behavior. Any sample in which the approach to the final fluorescence is substantially slower than in the above examples should be suspected of interference with the development reaction. For samples in which little or no fluorescence has developed, it may be impossible to assess the development kinetics.
4. Absolute certainty regarding interference with the SIRT2 Developer reaction can only be obtained through an assay in which the compound in question is tested for its effect on the reaction of Deacetylated Standard with the SIRT2 Developer. Using a standard curve such as that described in the previous section, determine the concentration of Deacetylated Standard that will yield a signal similar to that produced after development of a control (no inhibitor) SIRT2 reaction. Mix 40 µl of the Diluted Deacetylated Standard with 10 µl inhibitor or 10 µl Assay Buffer. Initiate development by adding 50 µl of 0.2X SIRT2 Developer to each well. Follow fluorescence development by reading at 1 or 2 min. intervals for 30 min If a test inhibitor sample reaches its final fluorescence significantly more slowly than the control then there may be interference with the SIRT2 Developer reaction. Compounds that decrease the final fluorescence signal without slowing the kinetics of its development may be quenching the fluorescence signal rather than interfering with the SIRT2 Developer reaction.
5. Once it is determined that a particular substance does interfere with the SIRT2 Developer reaction, it may be possible to adjust reaction conditions to eliminate this effect. In cases where the same final fluorescence is achieved, but more slowly than the control, simply extending the incubation time after addition of the SIRT2 Developer would be sufficient. Other possible adjustments include increasing the volume of SIRT2 Developer WS used per well (e.g. to 100 µl). Both approaches may be used separately or in combination.

Table 2: Assay Mixtures for Test Compound Retesting With Deacetylated Standard

The appropriate dilution of the Deacetylated Standard, in Assay Buffer may be determined from the standard curve and should be the concentration producing a fluorescent signal equal to that produced by control (no Test Compound) samples in the SIRT2 assay. The dilution in Assay Buffer is prepared at 1.25X this concentration to compensate for the 4/5 dilution due to addition of 10 µl of Assay Buffer or 5X Test Compound.
Example data

Figure 4: Inhibitors of SIRT2

Initial deacetylation rates of SIRT2 were determined at 25 µM SIRT2, 25 µM NAD+ (37°C) in the presence of the indicated concentrations of suramin or nicotinamide. Reactions were stopped with SIRT2 Develope WS and fluorescence measured (Ex. 360 nm, Em. 460 nm, gain=85).

Figure 5: Dependence of SIRT2 Kinetics on the Concentration of SIRT2

Initial deacetylation rates of SIRT2 were determined with 10 min incubations (37°C) in the presence of 3 mM NAD+. Reactions were stopped with SIRT2 Developer WS and fluorescence measured (Ex. 360 nm, Em. 460 nm, gain=85). Each point represents the mean of three determinations and the error bars are standard errors. The line is a non-linear least squares fit to the Michaelis-Menten equation. The Km for SIRT2 was 186 µM and the Vmax was 3510 AFU/min.

Figure 6: Dependence of SIRT2 Kinetics on the Concentration of NAD+

Initial deacetylation rates of SIRT2 were determined with 20 min. incubations (37°C) in the presence of 1 mM SIRT2. Reactions were stopped with SIRT2 Develope WS and fluorescence measured (Ex. 360 nm, Em. 460 nm, gain=85). Each point represents the mean of three determinations and the error bars are standard errors. The line is a non-linear least squares fit to the Michaelis-Menten equation. The Km for NAD+ was 547 µM and the Vmax was 4931 AFU/min.
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