SIRT5 Protein, Human (Flag)

SIRT5 Proteins, belonging to the sirtuin family, possess a sirtuin core domain and share homology with yeast Sir2 protein. They are classified in class III of the sirtuin family and undergo alternative splicing, resulting in multiple transcript variants. Their ubiquitous expression in tissues like the heart, liver, and 25 others suggests their involvement in diverse cellular processes. SIRT5 Protein, Human (Flag) is the recombinant human-derived SIRT5 protein, expressed by E. coli , with N-Flag labeled tag.

SIRT5, a member of the sirtuin family, shares homology with the yeast Sir2 protein and is characterized by a sirtuin core domain. The sirtuin family is divided into four classes, and while the specific functions of human sirtuins are yet to be fully elucidated, yeast sirtuin proteins are known to regulate epigenetic gene silencing and suppress recombination of rDNA. Studies suggest that human sirtuins may serve as intracellular regulatory proteins with mono-ADP-ribosyltransferase activity. SIRT5, classified within class III of the sirtuin family, undergoes alternative splicing, resulting in multiple transcript variants. The expression of SIRT5 is ubiquitous, observed in various tissues, including the heart, liver, and 25 other tissues, highlighting its likely involvement in diverse cellular processes.

Measured by its ability to remove the succinyl group from 25 μM fluorogenic peptide substrate incubate at room temperature for 30 minutes. The specific activity is 181.22 pmol/min/μg.

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  Western blot analysis of protein Human SIRT5 (lane 2(10ng) and lane 3(20ng)) and Human BRD4 (HY-P7846) (lane 4(10ng) and lane 5(20ng)) using Flag Tag (HRP) (HY-P80111A) Mouse mAb. Proteins were transferred to a PVDF membrane and blocked with 5% non-fat milk in TBST for 2 hour at room temperature. The primary antibody (1/1000) in 5% non-fat milk in TBST at 4°C overnight.

Materials
Assay buffer: 25 mM Tris, 150 mM NaCl, 1 mM DTT, pH 8.0
TCNB buffer：50 mM Tris, 0.15 M NaCl, 10 mM CaCl₂, 0.05% Brij-35 (w/v), pH 7.5 (TCNB)
PRSS3/Trypsin-3 Protein, Human (HEK293, His) (HY-P75985)
Activator Protein: Enterokinase Protein, Bovine (His)
Stop Solution: 8 ng/μL Trypsin, 4 mM Nicotinamide, 50 mM Tris, 100 mM NaCl, 30% (v/v) Isopropanol, pH 8.0
Beta-NAD: Dilute in ultrapure water to 100 mM
Substrate: FLUOR DE LYS®-Succinyl, Desuccinylase Substrate (5 mM)
Standard: 7-Amino-4-methylcoumarin (HY-D0027)

Procedure
1. Prepare a standard curve using AMC standards with concentrations of 10000, 5000, 2500, 1250, 625, 312.5, 156.25, 78.125, 19.53, 9.76, 0 pmol. Read the fluorescence at excitation and emission wavelengths of 380 nm and 460 nm, respectively.
2. Activation of Human Trypsin-3:
a. Dilute Bovine Enterokinase to 2 μg/mL in TCNB buffer;
b. Dilute Human Trypsin-3 to 200 μg/mL in TCNB buffer;
c. Mix equal volumes of the diluted proteins;
d. Incubate overnight at room temperature.
3. Dilute the Human SIRT5 to 10 μg/mL in assay buffer.
4. Dilute the substrate to 100 μM in assay buffer containing 2 mM beta-NAD.
5. Add 25 μL of 10 μg/mL Human SIRT5 and 25 μL of diluted substrate to the wells. For the control group, add only 25 μL of 10 μg/mL Human SIRT5.
6. Seal the plate and incubate at 37°C for 30 minutes.
7. Add 50 μL of stop solution to all wells and mix. For the control wells, add the stop solution first and then add 25 μL of substrate.
8. Seal the plate and incubate at room temperature for 15 minutes.
9. Read the fluorescence in endpoint mode at excitation and emission wavelengths of 380 nm and 460 nm, respectively.
10. Calculate specific activity:

Human

E. coli

N-Flag

NP_036373.1/Q9NXA8-1 (R2-S310)

23408  [NCBI]

Full Length of Isoform-1

RPLQIVPSRLISQLYCGLKPPASTRNQICLKMARPSSSMADFRKFFAKAKHIVIISGAGVSAESGVPTFRGAGGYWRKWQAQDLATPLAFAHNPSRVWEFYHYRREVMGSKEPNAGHRAIAECETRLGKQGRRVVVITQNIDELHRKAGTKNLLEIHGSLFKTRCTSCGVVAENYKSPICPALSGKGAPEPGTQDASIPVEKLPRCEEAGCGGLLRPHVVWFGENLDPAILEEVDRELAHCDLCLVVGTSSVVYPAAMFAPQVAARGVPVAEFNTETTPATNRFRFHFQGPCGTTLPEALACHENETVS

Approximately 33-37 kDa

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  Greater than 95% as determined by reducing SDS-PAGE.

Lyophilized powder.

Lyophilized from a 0.2 μm filtered solution of PBS, 1 mM DTT, pH 8.2 or 20 mM PB, 150 mM NaCl, 1 mM DTT, pH 7.8 or PBS, pH 7.8, 1mM DTT, 5% trehalose, 5% mannitol and 0.01% Tween80 or PBS, pH 7.8, 8% trehalose.

<1 EU/μg, determined by LAL method.

It is not recommended to reconstitute to a concentration less than 100 μg/mL in ddH₂O.

Stored at -20°C for 2 years from date of receipt. After reconstitution, it is stable at 4°C for 1 week or -20°C for longer (with carrier protein). It is recommended to freeze aliquots at -20°C or -80°C for extended storage.

Room temperature in continental US; may vary elsewhere.