Recombinant Human PSAT1 His-tag Protein, 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 MES and NaCl.|
|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, 16 mM Ammonium Acetate, pH 8.5
- Recombinant Human PSAT1 (rhPSAT1) (Catalog # 10342-PS)
- Recombinant Human PHGDH (rhPHGDH) (Catalog # 10131-DH)
- O-Phospho-L-serine (Tocris,
100 mM stock in 20 mM Tris, pH 8.5
- beta -Nicotinamide Adenine Dinucleotide, reduced (NADH) (Sigma, Catalog # N8129), 20 mM stock in 0.1 M Sodium Borate, pH 9.0
- alpha -Ketoglutaric acid (Sigma, Catalog # K2010), 1 M stock in deionized water
- Pyridoxal 5'-phosphate (Sigma, Catalog # P9255), 100 mM stock in 1 M HEPES, pH 8.0
- 96-well Clear Plate (Catalog # DY990)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Prepare a substrate mixture containing 0.4 mM NADH, 2 mM alpha -Ketoglutaric acid, 40 µM Pyridoxal 5'-phosphate, 2 mM O-Phospho-L-serine and 14 µg/mL rhPHGDH in Assay Buffer. Mix well and incubate at room temperature for 5 minutes.
- Dilute rhPSAT-1 to 20 µg/mL in Assay Buffer.
- Load 50 µL of 20 µg/mL rhPSAT-1 into wells of a plate, 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 an absorbance of 340 nm in kinetic mode for 10 minutes with a lag time of 3 minutes.
- Calculate specific activity:
Specific Activity (pmol/min/µg) =
|Adjusted Vmax* (OD/min) x well volume (L) x 1012 pmol/mol x (-1)|
|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
- rhPSAT-1: 1.0 µg
- O-Phospho-L-serine: 1 mM
- NADH: 0.2 mM
- alpha -Ketoglutaric acid: 1 mM
- Pyridoxal 5'-phosphate: 20 µM
- rhPHGDH: 0.7 µg
Recombinant Human PSAT1 His-tag (Catalog # 10342-PS) is measured by its ability to produce 3-phosphooxypyruvate.
2 μg/lane of Recombinant Human PSAT1 His-tag (Catalog # 10342-PS) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing a band at 40 kDa under reducing conditions.
Phosphoserine aminotransferase (PSAT1), a pyridoxal-phosphate (PLP)-dependent cytosolic protein catalyzes the conversion of 3-phosphohydroxypyruvate into phosphoserine; the reversible second step in the de novo serine synthesis pathway. PSAT1 forms an active a/b domain structure homodimer where each monomer is 40 kDa and 370 amino acids (1). PSAT1 contains a catalytic lysine and bound PLP in the active site. Although serine is considered a nonessential amino acid, de novo synthesis is essential in the brain due to insufficient delivery through the blood brain barrier. PSAT1 expression is concentrated in brain, liver, kidney, and pancreas suggesting these tissues are primary sites for de novo serine synthesis (2). Mutations in PSAT1 can cause Neu-Laxova syndrome, a serine-deficiency disorder (3). PSAT1 has been reported to signal through the GSK3beta/beta-catenin pathway (4, 5), regulate alpha keto-glutarate in a role essential for embryonic stem cell self-renewal and pluripotency (6) and promote migratory metastasis and proliferation in roles independent of serine synthesis (7,8). PSAT1 is up-regulated and associated with poor prognosis in several cancers including nasopharyngeal (9), breast (10), and esophageal squamous carcinoma (11). PSAT1 has been proposed as a promising target for tumor suppression in colorectal, esophageal, and breast cancers (8, 12, 13). It has been proposed as a target in brain tumors through a novel stabilization mechanism (14) and for inhibition of the serine production pathway in tuberculosis (15).
- Singh, R. S. et al. (2019) Int. J. Biol. Macromol. 132:1012.
- Baek, J. Y. et al. (2003) Biochem. J. 373:191.
- Acuna-Hidalgo, R. et al. (2014) Am. J. Hum. Genet. 95:285.
- Gao, S. et al. (2017) J. Exp. Clin. Cancer Res. 36:179.
- Dai, J. et al. (2019) J. Transl. Med. 17:190.
- Hwang, I. Y. et al. (2016) Cell Metab. 24:494.
- Yang, Y. et al. (2015) Int. J. Cancer 136:E39.
- Metcalf, S. et al. (2019) Clin. Exp. Metastasis. [epub ahead of print]
- Liao, K. M. et al. (2016) J. Cancer 7:1088.
- De Marchi, T. et al. (2017) Sci. Rep. 7:2099.
- Liu, B. et al. (2016) Cell Physiol. Biochem. 39:395.
- Yang, Y. et al. (2018) Cancer Manag. Res. 10:6537.
- Sun, C. et al. (2018) Cancer Manag. Res. 10:4581.
- Jiang, J. et al. (2019) Autophagy. [epub ahead of print]
- Haufroid, M. and Wouters, J. (2019) Pharmaceuticals 12:E66.
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