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Recombinant Mouse Acetylcholinesterase/ACHE Protein, CF

Catalog #: 5518-CE Datasheet / COA / SDS
Catalog # Availability Size / Price Qty
5518-CE-010
R&D Systems Recombinant Proteins and Enzymes
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Recombinant Mouse Acetylcholinesterase/ACHE Protein, CF Summary

Product Specifications

Purity
>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
Activity
Measured by its ability to cleave Acetylthiocholine. The specific activity is >250 nmol/min/μg, as measured under the described conditions.
Source
Mouse myeloma cell line, NS0-derived mouse Acetylcholinesterase/ACHE protein
Glu32-Leu614, with a C-terminal 10-His tag
Accession #
P21836
N-terminal Sequence
Analysis
Glu32
Predicted Molecular Mass
66 kDa
SDS-PAGE
68-73 kDa, reducing conditions

Product Datasheets

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5518-CE

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.

5518-CE

Formulation Supplied as a 0.2 μm filtered solution in Sodium Acetate and NaCl.
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.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.

Assay Procedure

Materials
  • Assay Buffer: 100 mM Sodium Phosphate, 0.05% (w/v) Brij-35, pH 7.5
  • Recombinant Mouse Acetylcholinesterase/ACHE (rmACHE) (Catalog # 5518-CE)
  • Substrate: Acetylthiocholine (ATC) (Sigma Catalog # A5626), 20 mM stock in DMSO
  • 5,5’-dithio-bis (2-nitrobenzoic acid) (DTNB) (Sigma Catalog # D-8130), 10 mM stock in DMSO
  • 96 well Clear Plate (Costar, Catalog #  92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rmACHE to 0.002 µg/mL in assay buffer.
  2. Combine equal volumes of stock DTNB and ATC to form substrate mixture.
  3. Dilute substrate mixture to a final concentration of 200 µM ATC and 100 µM DTNB in diH2O.
  4. Load into a 96 well clear plate 50 µL the diluted rmACHE.  For a blank, load 50 µL of the assay buffer.
  5. Start the reaction by adding 50 µL of the ATC/DTNB mixture to wells.
  6. Read in kinetic mode for 5 minutes at an absorbance of 405 nm.
  7. Calculate specific activity:

     Specific Activity (nmol/min/µg) =

 Adjusted Vmax* (OD/min) x well volume (L) x 109 nmol/M
ext. coeff** (M-1cm-1) x path corr.*** (cm) x amount of enzyme (µg)

     *Adjusted for Substrate Blank
     **Using the extinction coefficient 13260 M-1cm-1
     ***Using the path correction 0.32 cm
     Note: the output of many spectrophotometers is in mOD Per Well:
  • rmACHE: 0.0001 µg
  • DTNB: 50 µM
  • ATC: 100 µM
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: Acetylcholinesterase/ACHE

The classical role of ACHE is to terminate cholinergic neurotransmission by hydrolysis of acetylcholine (ACH) (1). ACHE is thought to be involved in the pathology of Alzheimer's disease (AD) by accelerating the assembly of A beta peptides into fibrillar species through forming complexes with A beta via the peripheral anionic site on ACHE. ACHE inhibitors have been used to delay symptoms of AD patients by virtue of their ability to enhance ACH availability, as well as reduce amyloidogenesis and subsequent neurotoxicity (2). Its involvement in the cholinergic anti-inflammatory pathway connects ACHE with a possible marker of low-grade systemic inflammation in obesity, hypertension, coronary heart disease, and AD (3). Alternative splicing produces three isoforms: an amphipathic form that exists as both monomeric and mutimeric forms, a soluble-monomeric form lacking the cysteine residue near the C-terminus, and a GPI-anchored dimeric form found in the membranes of erythrocytes (1). The recombinant mouse ACHE (rmACHE) was expressed as the amphipathic form that consists of soluble monomer and mutimeric forms.

References
  1. Grisaru, D. et al. (1999) Eur. J. Biochem, 264:672.
  2. Campbell, V. A. and Gowran, A. (2007) Br. J. Pharm. 152:655. 
  3. Das, U. N. (2007) Med. Sci. Monit. 13:RA214.
Entrez Gene IDs
43 (Human); 11423 (Mouse); 83817 (Rat)
Alternate Names
acetylcholinesterase (Yt blood group); Acetylcholinesterase; ACHE; apoptosis-related acetylcholinesterase; ARACHE; EC 3.1.1; EC 3.1.1.7; N-ACHE; Yt blood group; YT

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