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Recombinant Human Enolase 2 His-tag Protein, CF

Catalog #: 10412-EN Datasheet / COA / SDS
Catalog # Availability Size / Price Qty
10412-EN-050
Recombinant Human Enolase 2 His-tag Protein Binding Activity
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Recombinant Human Enolase 2 His-tag Protein, CF Summary

Product Specifications

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.
Activity
Measured by its ability to convert phosphoglyceric acid to phosphoenolpyruvate.
The specific activity is >6,000 pmol/min/μg, as measured under the described conditions.
Source
E. coli-derived human Enolase 2/Neuron-specific Enolase protein
Met1-Leu434
with an N-terminal Met and 6-His tag
Accession #
P09104.3
N-terminal Sequence
Analysis
Met
Predicted Molecular Mass
48 kDa
SDS-PAGE
46 kDa, under reducing conditions

Product Datasheets

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10412-EN

Product Datasheet
COA
SDS

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.

10412-EN

Formulation Supplied as a 0.2 μm filtered solution in MES, NaCl, KCl and MgSO4.
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, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.

Assay Procedure

Materials
  • Assay Buffer: 50 mM Tris, 30 mM MgCl2, 200 mM KCl, pH 7.5
  • Recombinant Human Enolase 2/Neuron‑specific Enolase His-tag (rhENO-2) (Catalog # 10412-EN).
  • Substrate: L-2-Phosphoglyceric acid (2-PG) (Sigma, Catalog # 19710), 100 mM stock in deionized water
  • Recombinant Human PKM-2 (PKM-2) (Catalog # 7244-PK)
  • Recombinant Human LDH-A (LDH-A) (Catalog # 9158-HA)
  • Adenosine diphosphate (ADP) (Sigma, Catalog # A2754), 200 mM stock in deionized water
  • beta -Nicotinamide adenine dinucleotide, reduced disodium salt hydrate ( beta -NADH) (Sigma, Catalog # N8129), 20 mM stock in 0.1 M Sodium Borate, pH 9.0
  • 96-well clear Plate (Catalog # DY990)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Prepare Reaction Mixture containing 20 µg/mL rhPKM-2, 5 µg/mL rhLDH-A, 14 mM ADP, 800 µM beta -NADH, and 8 mM 2-PG in Assay Buffer.
  2. Incubate Reaction Mixture at room temperature for 5 minutes.
  3. Dilute rhENO-2 to 1 µg/mL in Assay Buffer.
  4. In a plate, load 50 µL of 1 µg/mL rhENO-2, and start the reaction by adding 50 µL of Reaction Mixture. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of Reaction Mixture.
  5. Read at an absorbance of 340 nm in kinetic mode for 10 minutes with a lag time of 3 minutes.
  6. 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

Per Well:
  • rhENO-2: 0.05 µg
  • 2-PG: 4 mM
  • ADP: 7 mM
  • beta -NADH: 400 µM














Scientific Data

Binding Activity Recombinant Human Enolase 2 His-tag Protein Binding Activity View Larger

2 μg/lane of Recombinant Human Enolase 2/Neuron-specific Enolase His-tag (Catalog # 10412-EN) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing a band at 46 kDa under reducing conditions.

Reconstitution Calculator

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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Background: Enolase 2/Neuron-specific Enolase

Neuron specific enolase (NSE), also known as ENO2 or gamma-enolase, is a dimeric, Mg2+-dependent enzyme that catalyzes the dehydration of 2-phospho-D glycate (PGA) to phosphoenolpyruvate (PEP) in the glycolytic pathway and catalyzes the reverse reaction in gluconeogenesis. There are three major isozymes of enolase expressed in selective vertebrate tissues from separate genes: alpha (ENO1), beta (ENO3), and gamma (ENO2). NSE is a highly expressed, specific neuron isozyme (1) making it a useful marker for tumors derived from neuronal cells. Neuron-specific enolase is implicated as a diagnostic and prognostic marker in numerous diseases including early small cell lung cancer (2), prostate cancer (3), multiple myeloma (4), traumatic brain injury (5), acute spinal cord injury (6), acute ischemic stroke (7), and post-concussion symptoms (8).  NSE expression and activity are increased in neuronal and glial activation and injury (9), risk factors implicated in neurodegenerative disease. Elevation of NSE promotes glycolysis, proliferation, activation and migration through its C-terminus to activate PI3K and MAPK signal transduction pathways (6, 10) while inhibition of enolase has been shown to attenuate inflammatory events (11, 12). NSE can be regulated through cleavage of the C-termini by cathepsin X (13, 14) or inhibited directly by antibiotic SF2312 (15). Inhibition has been proposed as a therapeutic strategy in cancer (16).

References
  1. Marangos, P.J. and D.E. Schmechel, (1987) Annu. Rev. Neurosci. 10:269.
  2. Huang, L. et al. (2017) Oncotarget 38:64358.
  3. Muoio, B. et al. (2018) Int. J. Markers 33:10.
  4. Yang, H. et al. (2014) PLoS One. 9:e94304.
  5. Cheng, F. et al. (2014) PLoS One 9:e106680.
  6. Haque, A. et al. (2016) Metab. Brain Dis. 31:487.
  7. Anand, N. et al. (2005) Cerebrovasc. Dis. 20:213.
  8. Mercier, E. et al. (2018) Brain Inj. 32:29.
  9. Isgro, M.A. et al. (2015) Adv. Exp. Med. Biol. 867:125.
  10. Hafner, A. et al. (2013) Aging Cell 12:604.
  11. Polcyn, R. et al. (2017) J. Clin. Cell. Immunol. 8:536.
  12. Haque, A. et al. (2017) Neurochem. Res. 42:2777.
  13. Obermajer, N. et al. (2009) Int. J. Biochem. Cell Biol. 41:1685.
  14. Hafner, A. et al. (2012) Biochem. J. 443:439.
  15. Leonard, P.G. et al. (2016) Nat. Chem. Bio. 12:1053.
  16. Muller, F.L. et al. (2012) Nature. 488:337.
Entrez Gene IDs
2026 (Human); 13807 (Mouse); 24334 (Rat)
Alternate Names
2-phospho-D-glycerate hydrolyase; 2-phospho-D-glycerate hydro-lyase; EC 4.2.1.11; ENO2; enolase 2 (gamma, neuronal); Enolase 2; gamma-Enolase; Neural enolase; neuron specific gamma enolase; neurone-specific enolase; Neuronspecific Enolase; Neuron-specific enolase; NSE

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