Recombinant Human alpha-L-Iduronidase/IDUA His Protein, CF
Recombinant Human alpha-L-Iduronidase/IDUA His Protein, CF Summary
Product Specifications
Ala26-Pro653, with a C-terminal 10-His tag
Analysis
Product Datasheets
Carrier Free
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.
11180-GH
| Formulation | Supplied as a 0.2 μm filtered solution in Sodium Acetate, NaCl and Glycerol. |
| 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 Procedure
- Assay Buffer: 50 mM Sodium Acetate, 150 mM NaCl, 0.02% Brij-35 (w/v), pH 3.5
- Development Buffer: 0.1 M Tris, pH 9.0
- Recombinant Human IDUA (rhIDUA) (Catalog # 11180-GH)
- Substrate: 4-methylumberlliferyl-alpha -L-Iduronide, 20 mM stock in DMSO
- Black 96-well Plate
- Fluorescent Plate Reader
- Dilute rhIDUA to 0.2 µg/mL in Assay Buffer. Minimize the number of dilution steps to obtain the best activity results.
- Dilute Substrate to 800 µM in Assay Buffer.
- Combine equal volumes of 0.2 µg/mL rhIDUA and 800 µM Substrate. Include a Substrate Blank containing Assay Buffer and Substrate.
- Incubate reactions and Substrate Blank at room temperature for 10 minutes.
- Dilute mixtures to 0.005 µg/mL in Developing Buffer.
- Load 100 µL of the diluted mixtures into a plate.
- Read plate at excitation and emission wavelengths of 365 nm and 445 nm (top read), respectively, in endpoint mode.
- Calculate specific activity:
Specific Activity (pmol/min/µg) = | Adjusted Fluorescence* (RFU) x Conversion Factor** (pmol/RFU) |
| Incubation time (min) x amount of enzyme (µg) |
- rhIDUA: 0.0005 µg
- Substrate: 20 µM
Scientific Data
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2 μg/lane of Recombinant Human alpha-L-Iduronidase/IDUA His-tag Protein (Catalog # 11180-GH) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 80-88 kDa.
Reconstitution Calculator
Background: alpha-L-Iduronidase/IDUA
a-L-Iduronidase is a member of the glycoside hydrolase family encoded by the IDUA gene (1). It is an important enzyme required for the lysosomal degradation of glycosaminoglycans (GAGS) and hydrolyzes the non-reducing terminal a-L-iduronic acid residues in GAGS including dermatan sulfate and heparan sulfate. Human IDUA is a 653 aa protein composed of a signal peptide removed in the lysosome for mature form and three domains: a triosephosphate isomerase barrel fold containing the catalytic site, a B-sandwich domain, and an Ig(Ig)-like domain. The protein has six reported N-glycosylation sites and the glycosylation status of the enzyme correlates with its catalytic activity (1). More than 55-disease associated missense mutations in the IDUA gene have been identified (1). Mutations in IDUA that result in enzymatic deficiency lead to the autosomal recessive disease mucopolysaccharidosis type I (MPS I) (2). MPS I can be classified as three clinical subtypes; Hurler syndrome, Hurler-Scheie syndrome, and Scheie syndrome with decreasing severity, respectively. MPS I causes progressive cellular, tissue and organ damage, and several clinical studies using enzyme replacement therapy show positive results (3,4). More recently, the IDUA gene has been linked to osteoporosis (5,6).
- Maita, N. et al. (2013) Proc. Natl. Acad. Sci. 110:14628.
- Scott, H.S. et al. (1995) Hum. Mutat. 6:288.
- Wraith, J.E. (2005) Expert Opin. Pharmacother. 6:489.
- Jameson, E. (2016) Cochrane Database Syst. Rev. 4: CD009354.
- Kodric, K. et al. (2016) Wien Klin Wochenschr. 128:480.
- Niu, T. et al. (2016) J. Bone Miner. Res. 31:358.
FAQs
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