Recombinant Human PHGDH His-tag, CF Summary
with a C-terminal 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, 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.
- 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 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 extinction coefficient 6220 M-1cm-1.
***Using the path correction 0.32 cm.
- rhPHGDH: 0.25 μg
- PGA: 10 mM
- beta -NAD: 2 mM
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).
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.
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).
- Unterlass, J. E. et al. (2017) Oncotarget 8:104478.
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- 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.
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- Mattaini, K. R. et al. (2015) Cancer Metab. 3:5.
- Wang, Q. et al. (2016) Cell Chem. Biol. 24:1.
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- Unterlass, J. E. et al. (2018) Oncotarget 9:13139.
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