Recombinant Human Glutaminase His-tag Protein, CF

Catalog # Availability Size / Price Qty
10115-GL-020
Glutaminase Protein Enzyme Activity
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Product Details
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Recombinant Human Glutaminase His-tag Protein, CF Summary

Purity
>80%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Activity
Measured by its ability to hydrolyze glutamine to glutamate. The specific activity is >12000 pmol/min/μg, as measured under the described conditions.
Source
E. coli-derived human Glutaminase protein
Ser17-Leu669
with N-terminal Met and 6-His tag
Accession #
N-terminal Sequence
Analysis
Met
Predicted Molecular Mass
73 kDa
SDS-PAGE
59-74 kDa, under reducing conditions

Product Datasheets

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.

10115-GL

Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl, TCEP, Glycerol and CHAPS.
Shipping The product is shipped with polar packs. 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, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.

Assay Procedure

Materials
  • Assay Buffer: 100 mM Tris, 150 mM K2HPO4, pH 8.6
  • Recombinant Human Glutaminase Kidney Isoform His-tag (rhGLS) (Catalog # 10115-GL)
  • Glutamate dehydrogenase (GIDH) (Sigma, Catalog # G7882), 400 U/mL stock in 10 mM HEPES, pH 7.0
  • Nicotinamide adenine dinucleotide ( beta -NAD) (Sigma, Catalog # N6522), 100 mM stock in deionized water
  • L-Glutamine (Sigma, Catalog # G8540), 200 mM stock in deionized water
  • 96-well Clear Plate (Catalog # DY990)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhGLS to 0.5 µg/mL in Assay Buffer.
  2. Prepare Substrate Mixture containing 50 mM L-Glutamine, 60 U/mL GIDH and 4 mM beta -NAD in Assay Buffer.
  3. Load into a plate 50 µL of rhGLS, and start the reaction by adding 50 µL of Substrate Mixture.  Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of Substrate Mixture.
  4. Read plate at 340 nm (absorbance) in kinetic mode for 5 minutes.
  5. Calculate specific activity:
     

     Specific Activity (pmol/min/µg) =

Adjusted Vmax* (OD/min) x well volume (L) x 1012 pmol/mol
ext. coeff** (M-1cm-1) x path corr.*** (cm) x amount of enzyme (µg)


*Adjusted for Substrate Blank.
**Using the extinction coefficient 6220 M-1cm-1.
***Using the path correction 0.32 cm.
Note: the output of many spectrophotometers is in mOD. Per Well:
  • rhGLS: 0.025 µg
  • L-Glutamine: 25 mM
  • GIDH: 3 U
  • beta -NAD: 2 mM

Data Images

Enzyme Activity Enzyme Activity View Larger

Recombinant HumanGlutaminase His-tag (Catalog # 10115-GL) is measured by its ability tohydrolyze glutamine to glutamate. The activity (orange) is higher than thecompetitor's Glutaminase (green).

SDS-PAGE SDS-PAGE View Larger

1 μg/lane of Recombinant Human Glutaminase His-tag (Catalog # 10115-GL) and 1 μg/lane of competitor Human Glutaminase was resolved with 4-20% SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by silver staining.

Reconstitution Calculator

Reconstitution Calculator

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: Glutaminase

Glutaminase (GLS) catalyzes the synthesis of glutamate from glutamine. There are two mammalian isozymes of glutaminase: kidney-type encoded by the GLS gene, and liver-type encoded by the GLS2 gene. The two isozymes are known to have different localization, functionality, and immunological and molecular characteristics (1, 2). The GLS gene expresses two splice variants with a conserved catalytic domain (3): kidney-type glutaminase (KGA) and a C-terminal truncated variant known as glutaminase C. Both splice variants form active tetramers and are activated by phosphate (4). The kidney glutaminase isoform, GLS, catalyzes the first reaction in the primary pathway for renal catabolism of glutamine to glutamate. It contains a mitochondrial signaling peptide, a nuclear receptor box, a catalytic glutaminase domain, three ankyrin repeats and a KEN box (5).  GLS promotes respiration and ATP generation in the mitochondria by providing an alternative input to the citric acid cycle. GLS is upregulated in response to oncogenes (5-7) in several cancers (8, 9). Cancer cells with altered metabolism have been shown to depend on GLS activity and upregulated glutamine consumption (10) for growth in acute myeloid leukemina (11), breast cancer (12), colorectal cancer (13), kidney cancer (14), lung cancer (15), melanoma (16) and pancreatic cancer (17). Based on its role in cancer, GLS is a pharmaceutical target for cancer therapy (10, 12, 13, 18).

References
  1. Kovacevic, Z. et al. (1983) Physiol. Rev. 63:547.
  2. Marquez, J. et al. (2006) Neurochem. Int. 48:465.
  3. Ramachandran, S. et al. (2016) Oncotarget. 7:57943.
  4. Huang, Q. et al. (2018) J. Biol. Chem. 293:3535.
  5. Thangavelu, K. et al. (2012) Proc. Natl. Acad. Sci. 109:7705.
  6. Wise, D. et al. (2008) Proc. Natl. Adac. Sci. 105:18782.
  7. Gao, P. et al. (2009) Nature. 458:762.
  8. Cline, M. S. et al. (2013) Sci. Rep. 3:2652.
  9. Cancer Genome Atlas Research Network, et al. (2013) Nat. Genet. 45:1113.
  10. Katt, W. P. et al. (2017) Future Med. Chem. 9:223.
  11. Willems, L. et al. (2013) Blood 122:3521.
  12. Gross, M. I. et al. (2014) Mol. Cancer Ther. 13:890.
  13. Huang, F. et al. (2014) Int. J. Clin. Exp. Pathol. 7:1093.
  14. Shroff, E. H. et al. (2015) Proc. Natl. Acad. Sci. 112:6539.
  15. van den Heuvel, A. P. et al. (2012) Cancer Biol. Ther. 13:1185.
  16. Hernandez-Davies, J. E. et al. (2015) J. Trans. Med. 13:1.
  17. Son, J. et al. (2013) Nature. 496:101.
  18. Zimmermann, S.C. et al. (2018) J. Med. Chem. [Epub ahead of print].
Long Name
Glutaminase Kidney Isoform, Mitochondrial
Entrez Gene IDs
2744 (Human); 14660 (Mouse); 24398 (Rat)
Alternate Names
AAD20; EC 3.5.1.2; GLS; GLS1DKFZp686O15119; glutaminase C; glutaminase kidney isoform, mitochondrial; Glutaminase; glutaminase, phosphate-activated; K-Glutaminase; KIAA0838FLJ10358; L-Glutamine Amidohydrolase

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