Recombinant Human PHGDH His-tag, CF

Name: Recombinant Human PHGDH His-tag, CF
Brand: rnd
SKU: 10131-DH

Catalog # 10131-DH

Datasheet / COA / SDS

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Key Product Details

Source:	E. coli
Accession #:	O43175-1
Applications:	Enzyme Activity

View Full Technical Details for Recombinant Human PHGDH His-tag, CF

Product Specifications

Source	E. coli-derived human PHGDH protein Ala2-Phe533 with a C-terminal 6-His tag
Purity	>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Endotoxin level	<0.10 EU per 1 μg of the protein by the LAL method.
N-terminal sequence Analysis	Ala2
Predicted Molecular Mass	57 kDa
SDS-PAGE	57 kDa, under reducing conditions
Activity	Measured by the ability to catalyze the oxidation of 3-phospho-D-glycerate. The specific activity is >350 pmol/min/μg, as measured under the described conditions.

Recombinant Human PHGDH His-tag, CF Scientific Data Examples

	Recombinant Human PHGDH His-tag Enzyme Activity Recombinant Human PHGDH His-tag (Catalog # 10131-DH) is measured by its ability to catalyze the oxidation of 3-phospho-D-glycerate. The activity (orange) is over 2-fold than the competitor's PHGDH (green).
	Recombinant Human PHGDH His-tag SDS-PAGE 2 μg/lane of Recombinant Human PHGDH His-tag (Catalog # 10131-DH) and 2 μg/lane of competitor Human PHGDH was resolved with 4-20 % SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie.

Formulation, Preparation and Storage

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.

10131-DH

Formulation	Supplied as a 0.2 μm filtered solution in Tris, NaCl, Glycerol and TCEP.
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.

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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Assay Procedure

Materials

Assay Buffer: 50 mM Tris, 800 mM NaCl, 0.2 mM DTT, pH 9.0
Recombinant Human Phosphoglycerate Dehydrogenase (rhPHGDH) (Catalog # 10131-DH)
3-Phospho-D-Glyceric Acid (PGA) (Sigma, Catalog # P8877), 200 mM stock in deionized water
beta -Nicotinamide Adenine Dinucleotide ( beta -NAD) (Sigma, Catalog # N6522), 100 mM stock in deionized water
UV Plate (Costar, Catalog # 3635)
Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

Dilute rhPHGDH to 5 µg/mL in Assay Buffer.
Prepare Substrate Mixture containing 20 mM PGA and 4 mM beta -NAD in Assay Buffer.
Load into a plate 50 μL of 5 μg/mL rhPHGDH, and start the reaction by adding 50 μL of Substrate Mixture. Include a Substrate Blank containing 50 μL of 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 V_max* (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 extinction coefficient 6220 M^-1cm^-1.
***Using the path correction 0.32 cm.

Per Well:

rhPHGDH: 0.25 μg
PGA: 10 mM
beta -NAD: 2 mM

Background: PHGDH

Phosphoglycerate dehydrogenase (PHGDH) catalyzes the oxidation of 3-phospho-D-glycerate to 3-phosphooxypyruvate, which is the first step of the L-serine biosynthesis pathway. Human PHGDH is a Type I NADH-dependent enzyme that forms an active oligomer where each monomer is composed of 4 domains: a substrate-binding domain, a nucleotide-binding domain, and two regulatory domains: ASB (allosteric substrate binding) and ACT (Aspartate kinase, Chorismate mutase, and Tyr A)(1). Serine biosynthesis by PHGDH is the sole source of serine biosynthesis in mammals (2) and conditional knockout confirmed L-serine synthesis by PHGDH is the source of D-serine in the brain (3). PHGDH null mice are embryonic lethal (4) and mutations that lead to PHGDH deficiency have been reported in infantile, juvenile and adult onset phenotypes (5, 6). PHGDH is also suggested to play a role in several cancers including breast (7), cervical (8), melanoma (9), colon (10), pancreatic (11), liver (12), and kidney (13) through increased PHGDH expression and enhanced tumor cell proliferation. Levels of PHGDH correlate with patient survival and may be used as a prognostic factor (14). A mutation resulting in inactive PHGDH (15), cell line knockdown (8, 11), and treatment with enzymatic inhibitors in cancer cells (13) result in decreased proliferation. Given the correlation between high PHGDH and cancer and the results from indirect inhibition of PHGDH activity, PHGDH is a pharmaceutical target for cancer therapy (16-18).

Item	Value
References	Unterlass, J. E. et al. (2017) Oncotarget 8:104478. Mattaini, K. R. et al. (2016) J. Cell Biol. 214:249. Yang, J.H. et al. (2010) J. Biol. Chem. 285:41380. Yoshida, K. et al. (2004) J. Biol. Chem. 279:3573. Tabatabaie, L. et al. (2010) Mol. Gen. Metab. 99:256. van der Crabben, S. N. (2013) J. Inherit. Metab. 36:613. Possemato, R. et al. (2011) Nature 476:346. Jing, Z. et al. (2015) Cancer Biol. Ther. 16:541. Ou, Y. et al. (2015) J. Biol. Chem 290:457. Yoon, S. et al. (2015) Oncology 89:351. Song, Z. et al. (2018) Gene 642:43. Liu, S. et al. (2017) Hepatology 66:631. Yoshino, H. et al. (2017) Cancer Res. 77:6321. Antonov, A. et al. (2014) Oncotarget 5:11004. Mattaini, K. R. et al. (2015) Cancer Metab. 3:5. Wang, Q. et al. (2016) Cell Chem. Biol. 24:1. Ravez, S. et al. (2017) J. Med. Chem. 60:1591. Unterlass, J. E. et al. (2018) Oncotarget 9:13139.
Long Name	3-Phosphoglycerate Dehydrogenase
Entrez Gene IDs	26227 (Human); 236539 (Mouse); 58835 (Rat)
Alternate Names	3,3-phosphoglycerate dehydrogenase, 3-PGDH, 3PGDH, D-3-phosphoglycerate dehydrogenase, EC 1.1.1, EC 1.1.1.95, MGC3017, PDG, PDGH3, PGAD, PGD, PGDH, PHGDH, SERA, phosphoglycerate dehydrogenase