Recombinant Human Glutaminase His-tag Protein, CF Summary
with N-terminal Met and 6-His tag
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.
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.
|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.
- 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
- Dilute rhGLS to 0.5 µg/mL in Assay Buffer.
- Prepare Substrate Mixture containing 50 mM L-Glutamine, 60 U/mL GIDH and 4 mM beta -NAD in Assay Buffer.
- 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.
- Read plate at 340 nm (absorbance) in kinetic mode for 5 minutes.
- 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
Recombinant Human Glutaminase His-tag (Catalog # 10115-GL) is measured by its ability to hydrolyze glutamine to glutamate. The activity (orange) is higher than the competitor's Glutaminase (green).
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.
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).
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