Recombinant Human PGK-1 Protein, CF Summary
Met1-Ile417, with an C-terminal 10-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 and Glycerol.|
|Shipping||The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.|
|Stability & Storage:||Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
- Assay Diluent: deionized water
- Recombinant Human PGK1 (rhPGK1) (Catalog # 5455-PK)
- 50 mM KH2PO4, pH 7.0
- 100 mM MgSO4 in deionized water
- 1.0 M Glycine in deionized water
- 50 mM DL-Glyceraldehyde 3-Phosphate (GAP) (Sigma, Catalog # G5251) in deionized water
- 10 mM beta -Nicotinamide adenine dinucleotide ( beta -NAD) (Sigma, Catalog # N6522). Prepare 200 mM stock in deionized water
- 10 mM Adenosine 5’-Diphosphate (ADP) (Sigma, Catalog # A2754). Prepare 200 mM stock in deionized water. Note: ADP degrades to AMP which is an inhibitor of rhPGK1. Be sure to aliquot and store the stock at ≤-20 °C. Prepare fresh when necessary.
- 0.25 µg/µL Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) (Sigma, Catalog # G5537) in 50% Glycerol
- UV Plate, 96 well (Costar, Catalog # 3635)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Prepare substrate buffer by mixing the following (prepare fresh):
- 100 µL 50 mM KH2PO4, pH 7.0
- 20 µL 50 mM GAP
- 30 µL 10 mM beta -NAD
- 20 µL 10 mM ADP
- 50 µL 100 mM MgSO4
- 100 µL 1 M Glycine
- 20 µL 0.25 µg/µL GAPDH
- 160 µL deionized water
- Dilute rhPGK1 to 0.02 ng/µL in deionized water.
- Load in a 96 well UV plate 50 µL of the substrate buffer, and start the reaction by adding 50 µL of 0.02 ng/µL rhPGK1. Include a blank containing 50 µL of the substrate buffer and 50 µL deionized water.
- Read at 339 nm in kinetic mode for 5 minutes.
- Calculate specific activity:
Note: This amount will assay nine wells. If more volume is needed, multiply each component’s volume by the same number to get the desired amount.
Specific Activity (pmol/min/µg) =
|Adjusted Vmax* (OD/min) x well volume (L) x 1012 pmol/M|
|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 mODPer Well:
- rhPGK1: 0.001 μg
- Rxn mix: 5 mM KH2PO4, pH 7.0, 1 mM GAP, 0.3 mM beta -NAD, 0.2 mM ADP, 5 mM MgSO4, 100 mM Glycine, 5 ng/µL GAPDH
Phosphoglycerate kinase-1 (PGK-1) is a glycolytic enzyme that catalyzes the conversion of 1,3-diphosphoglycerate to 3 phosphoglycerate. The gene encoding PGK-1 is X-linked. Mutations of this gene may cause phosphoglycerate kinase deficiency, which is characterized by hemolytic anemia, muscle stiffness and mental retardation (1‑3). PGK 1 is induced by oxidative stress through the induction of hypoxia-inducible factor 1a and is a potential biomarker and therapeutic target for cancer (4‑7).
- Beutler, E. (2002) Br. J. Haematol. 136:3.
- Flanagan, J.M. et al. (2006) Br. J. Haematol. 134:233.
- Svaasand, E.K. et al. (2007) Muscle Nerve. 36:579.
- Hwang, T.L. et al. (2006) Proteomics. 6:2259.
- Wang, J. et al. (2007) Cancer Res. 67:149.
- Zieker, D. et al. (2008) Cell Physiol. Biochem. 21:429.
- Jang, C.H. et al. (2008) Biosci. Biotechnol. Biochem. 72:1799.
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