Recombinant Human Galactosylceramidase/GALC Protein, CF
Recombinant Human Galactosylceramidase/GALC Protein, CF Summary
Product Specifications
Met1-Arg685, with a C-terminal 6-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.
7310-GH
| Formulation | Supplied as a 0.2 μm filtered solution in MES, 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 Citrate, 125 mM NaCl, 0.5% Triton® X-100, pH 4.5
- Stop Solution: 0.5 M Glycine, 0.3 M NaOH (~pH 10.0)
- Recombinant Human Galactosylceramidase/GALC (rhGALC) (Catalog # 7310-GH)
- Substrate: 4-methylumbelliferyl-beta -D-galactopyranoside (Sigma, Catalog # M1633), 10 mM stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rhGALC to 0.06 ng/μL in Assay Buffer.
- Dilute Substrate to 1 mM in Assay Buffer.
- Load in a plate 25 μL of 0.06 ng/μL rhGALC, and start the reaction by adding 25 μL of 1 mM Substrate. Include a Substrate Blank containing 25 μL of Assay Buffer and 25 μL of 1 mM Substrate.
- Seal plate and incubate at 37 °C for 20 minutes.
- After incubation, stop the reactions by adding 50 μL of Stop Solution to each well.
- Read 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) |
*Adjusted for Substrate Blank
**Derived using calibration standard 4-methylumbelliferone (Sigma, Catalog # M1381).
- rhGALC: 0.0015 μg
- Substrate: 0.25 mM
Scientific Data
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Recombinant Human Galactosylceramidase/GALC Protein Enzyme Activity Diagram. Recombinant Human Galactosylceramidase/GALC Protein, CF (Catalog # 7310-GH) catalyzes the hydrolysis of beta -d-galactocerebroside to beta -d-galactose and ceramide.
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Recombinant Human Galactosylceramidase/GALC Protein, CF (Catalog # 7310-GH) is measured by its ability to cleave a fluorogenic substrate, 4-Methylumbelliferyl-beta -D-galactopyranoside.
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2 μg/lane of Recombinant Human Galactosylceramidase/GALC Protein (Catalog # 7310-GH) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 70-85 kDa.
Reconstitution Calculator
Background: Galactosylceramidase/GALC
Galactosylceramidase (GALC or galactocerebrosidase) is a lysosomal enzyme that hydrolyzes the galactose ester bonds of galactosylceramide, galactosylsphingosine, lactosylceramide, and monogalactosyldiglyceride, molecules that are important in the production of normal myelin (1). Mutations in the GALC gene have been associated with Krabbe’s Disease, also known as globoid cell leukodystrophy (2, 3, 4), a fatal autosomal recessive disorder resulting from the insufficient catabolism of these galactolipids. Krabbe’s Disease is morphologically characterized by almost total absence of myelin, severe gliosis, and the presence of characteristic multinucleated globoid cells in the white matter (4). In a recent study, cord blood transplants have been successful in stopping the disease (5).
- Chen, Y.Q. et al. (1993) Hum. Mol. Genet. 2:1841.
- Tappino, B. et al. (2010) Hum. Mutat. 31:E1894.
- Kodama, S. et al. (1982) J Neurochem. 39:1314.
- Suzuki, K. and Suzuki, Y. (1970) Proc. Natl. Acad. Sci. U.S.A. 66:302.
- Escolar, M.L. et al. (2005) N. Engl. J. Med. 352:2069.
Citations for Recombinant Human Galactosylceramidase/GALC Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
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Citations: Showing 1 - 3
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Substrate reduction therapy for Krabbe disease and metachromatic leukodystrophy using a novel ceramide galactosyltransferase inhibitor
Authors: MC Babcock, CR Mikulka, B Wang, S Chandriani, S Chandra, Y Xu, K Webster, Y Feng, HR Nelvagal, A Giaramita, BK Yip, M Lo, X Jiang, Q Chao, JC Woloszynek, Y Shen, S Bhagwat, MS Sands, BE Crawford
Scientific Reports, 2021-07-14;11(1):14486.
Species: Human
Sample Types: Recombinant Protein
Applications: Bioassay -
Glucocerebrosidases catalyze a transgalactosylation reaction that yields a newly identified brain sterol metabolite, galactosylated cholesterol
Authors: H Akiyama, M Ide, Y Nagatsuka, T Sayano, E Nakanishi, N Uemura, K Yuyama, Y Yamaguchi, H Kamiguchi, R Takahashi, JMFG Aerts, P Greimel, Y Hirabayash
J. Biol. Chem., 2020-03-06;0(0):.
Applications: Bioassay -
Fluorimetric assay with a novel substrate for quantification of galactocerebrosidase activity in dried blood spot specimens
Authors: AJ Ullal, H Pham, R Singh, P Ross, CA Graham, SM Norton, MH Nuffer, DS Burns, AE Eckhardt, M Escolar, D Bali, VK Pamula
Pract Lab Med, 2019-10-16;18(0):e00141.
Species: Human
Sample Types: Protein
Applications: Bioassay
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