Recombinant Human Sirtuin 2/SIRT2 Protein, CF

Catalog #: 4358-DA Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
4358-DA-050

R&D Systems Recombinant Proteins and Enzymes

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Recombinant Human Sirtuin 2/SIRT2 Protein, CF Summary

Product Specifications

Purity

>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

Activity

Measured by its ability to remove the acetyl group from a fluorogenic peptide substrate Ac-RGK(Ac)-AMC (Catalog # ES016) in a coupled assay. The specific activity is >3 pmol/min/µg, as measured under the described conditions.

Source

Spodoptera frugiperda, Sf 21 (baculovirus)-derived human Sirtuin 2/SIRT2 protein
Met1-Gln389, with a C-terminal 6-His tag

Accession #

Q8IXJ6

N-terminal Sequence
Analysis

No results obtained: Presumed blocked

Predicted Molecular Mass

44 kDa

SDS-PAGE

43-49 kDa, reducing conditions

Product Datasheets

4358-DA

Product Datasheet

COA

SDS

Carrier Free

What does CF mean?

CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.

What formulation is right for me?

In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.

4358-DA

Formulation	Supplied as a 0.2 μm filtered solution in Tris and Glycerol.
Shipping	The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage:	Use a manual defrost freezer and avoid repeated freeze-thaw cycles. 6 months from date of receipt, -20 to -70 °C as supplied. 3 months, -20 to -70 °C under sterile conditions after opening.

Assay Procedure

Materials

Assay Buffer: 25 mM Tris, 150 mM NaCl, 1 mM DTT, pH 8.0
Stop Solution: 8 ng/µL Recombinant Human Active Trypsin 3/PRSS3 (Catalog # 3714-SE), 4 mM Nicotinamide (Sigma, Catalog # 72340), 50 mM Tris, 100 mM NaCl, 30% (v/v) isopropanol, pH 8.0
Recombinant Human Sirtuin 2/SIRT2 (rhSIRT2) (Catalog # 4358-DA)
beta -Nicotinamide adenine dinucleotide hydrate ( beta -NAD) (Sigma, Catalog # N6522), 100 mM stock in diH₂O
Substrate: Ac-Arg-Gly-Lys(Ac)-AMC (Catalog # ES016), 20 mM stock in DMSO
F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

Dilute rhSIRT2 to 0.04 µg/µL in Assay Buffer.
Dilute Substrate to 500 µM in Assay Buffer containing 2 mM beta -NAD.
Mix 25 µLof 0.04 µg/µL rhSIRT2 and 25 µL of Substrate in plate. Add 25 µL of 0.04 µg/µL rhSIRT2 alone for the control.
Cover with parafilm or a plate sealer and incubate at 37 °C for 30 minutes.
Add 50 µL of Stop Solution to all wells. Mix. For control well(s), add 25 µL of Substrate after addition of Stop Solution.
Seal tightly and incubate at room temperature for 15 minutes.
Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively in endpoint mode.
Calculate specific activity:

Specific Activity (pmol/min/µg) =	Adjusted Fluorescence* (RFU) x Conversion Factor** (pmol/RFU)
	Incubation time (min) x amount of enzyme (µg)

*Adjusted for Control

**Derived using calibration standard 7-amino, 4-Methyl Coumarin (AMC) (Sigma, Catalog # A-9891).

Per Well:

rhSIRT2: 1 µg
Substrate: 125 µM

Reconstitution Calculator

Background: Sirtuin 2/SIRT2

Sirtuin 2, encoded by the SIRT2 gene, is also known as SIR2 (silent information regulator 2)-like protein 2. It is a nicotinamide adenine dinucleotide (NAD)-dependent histone/protein deacetylase (1, 2). The SIR2 family of enzymes is classified as class III histone deacetylases (HDACs) and has been implicated in many cellular processes that include histone deacetylation, gene silencing, chromosomal stability, and aging (3, 4). Unlike class I and class II HDACs, the enzymatic activity of class III HDACs is NAD dependent and insensitive to HDAC inhibitor trichostatin A (5).

References