Recombinant Human Glucosylceramidase/GBA Protein, CF

Catalog # Availability Size / Price Qty
7410-GHB-020
7410-GHB-01M
Product Details
Citations (6)
FAQs
Supplemental Products
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Recombinant Human Glucosylceramidase/GBA Protein, CF Summary

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 hydrolyze 4-methylumbelliferyl-beta -D-glucopyranoside. The specific activity is >200 pmol/min/μg, as measured under the described conditions.
Source
Chinese Hamster Ovary cell line, CHO-derived human Glucosylceramidase/GBA protein
Ala40-Gln536, with a C-terminal 6-His tag
Accession #
N-terminal Sequence
Analysis
Ala40
Predicted Molecular Mass
56 kDa
SDS-PAGE
60-75 kDa, reducing conditions

Product Datasheets

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.

7410-GHB

Formulation Supplied as a 0.2 μm filtered solution in Sodium Citrate.
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 Sodium Citrate, 25 mM Sodium Cholate, 5 mM DTT, pH 6.0
  • Stop solution: 0.5 M Glycine, 0.3 M NaOH (~pH 10)
  • Recombinant Human Glucosylceramidase/GBA (rhGBA) (Catalog # 7410-GHB)
  • Substrate: 4-Methylumbelliferyl-beta -D-glucopyranoside (Sigma, Catalog # M3633), 10 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhGBA to 0.2 ng/µL in Assay Buffer.
  2. Dilute Substrate to 6 mM in Assay Buffer.
  3. Load 25 µL of 0 2 ng/µL rhGBA into wells of a plate, and start the reactions by adding 25 μL of 6 mM Substrate. Include a Substrate Blank containing 25 µL of Assay Buffer and 25 µL of 6 mM Substrate.
  4. Seal plate and incubate at 37 °C for 20 minutes.
  5. After incubation, stop the reactions by adding 50 µL of Stop Solution to each well.
  6. Read at excitation and emission wavelengths of 365 nm and 445 nm (top read), respectively, in endpoint mode.
  7. 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 4methylumbelliferone (Sigma, Catalog # 69580).

Per Well:
  • rhGBA: 0.005 µg
  • Substrate: 1.5 mM
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: Glucosylceramidase/GBA

Glucosylceramidase is a lysosomal enzyme that cleaves the beta-glucosidic linkage of glucosylceramide (1, 2), an intermediate in glycolipid metabolism. The mature enzyme has 497 amino acids with a molecular weight of 62 kDa (3). Glycosylation occurs at four of five N-glycosylation sites and is essential for the trafficking and activity of the enzyme (4). The enzyme is activated in lysosomes by saposin C, although the mechanism of activation is not well understood (5). Defects in Glucosylceramidase are the cause of Gaucher disease, also known as glucocerebrosidase deficiency (6). Gaucher disease is the most prevalent lysosomal storage disease, characterized by accumulation of glucosylceramide in the reticulo-endothelial system. Symptoms of Gaucher disease may include enlarged spleen and liver, liver malfunction, skeletal disorders and bone lesions, severe neurologic complications, swelling of lymph nodes, anemia, low blood platelets and yellow fatty deposits on the white of the eye (7). Currently, enzyme replacement therapy is used to treat patients with the disease (8, 9).

References
  1. Sorge, J. et al. (1985) Proc. Natl. Acad. Sci. USA 82:7289.
  2. Ginns, E. I. et al. (1984) Biochem. Biophyl. Res. Commun. 123:574.
  3. Horowitz, M. et al. (1989) Genomics 4:87.
  4. Grace, M.E. et al. (1994) J. Biol. Chem. 269:2283.
  5. Bruhn, h. (2005) Biochem. J. 389:249.
  6. Liou, B. et al. (2006) J. Biol. Chem. 281:4242.
  7. Grabowski, G.A. (2008). Lancet 372:1263.
  8. Zheng, W. et al. (2007) Proc. Natl. Acad. Sci. USA 104:13192.
  9. Beutler, E. and Gelbart, T. (1996) Hum. Mutat. 8:207.
Entrez Gene IDs
2629 (Human); 14466 (Mouse)
Alternate Names
Acid beta-glucosidase; Alglucerase; Beta-glucocerebrosidase; D-glucosyl-N-acylsphingosine glucohydrolase; EC 3.2.1.45; GBA; GBA1; GC; GCB; GLUC; glucosidase, beta, acid; glucosidase, beta; acid (includes glucosylceramidase); Glucosylceramidase; Imiglucerase; lysosomal glucocerebrosidase

Citations for Recombinant Human Glucosylceramidase/GBA 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.

6 Citations: Showing 1 - 6
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  1. Mitochondrial dysfunction and mitophagy defect triggered by heterozygous GBA mutations
    Authors: H Li, A Ham, TC Ma, SH Kuo, E Kanter, D Kim, HS Ko, Y Quan, SP Sardi, A Li, O Arancio, UJ Kang, D Sulzer, G Tang
    Autophagy, 2019;15(1):113-130.
    Species: Mouse
    Sample Types: Tissue Homogenates
  2. Targeted delivery of lysosomal enzymes to the endocytic compartment in human cells using engineered extracellular vesicles
    Authors: MA Do, D Levy, A Brown, G Marriott, B Lu
    Sci Rep, 2019;9(1):17274.
    Species: N/A
    Sample Types: Protein
    Applications: Enzyme Assay
  3. ?-Synuclein accumulation and GBA deficiency due to L444P GBA mutation contributes to MPTP-induced parkinsonism
    Authors: SP Yun, D Kim, S Kim, S Kim, SS Karuppagou, SH Kwon, S Lee, TI Kam, S Lee, S Ham, JH Park, VL Dawson, TM Dawson, Y Lee, HS Ko
    Mol Neurodegener, 2018;13(1):1.
    Species: Mouse
    Sample Types: Whole Cells
    Applications: Bioassay
  4. Fluorescence-Quenched Substrates for Quantitative Live Cell Imaging of Glucocerebrosidase Activity
    Authors: RA Ashmus, DL Shen, DJ Vocadlo
    Meth. Enzymol., 2018;598(0):199-215.
    Species: Human
    Sample Types: Fluorescent Probe
    Applications: Bioassay
  5. Lysosomal Enzyme Glucocerebrosidase Protects against Abeta1-42 Oligomer-Induced Neurotoxicity.
    Authors: Choi S, Kim D, Kam T, Yun S, Kim S, Park H, Hwang H, Pletnikova O, Troncoso J, Dawson V, Dawson T, Ko H
    PLoS ONE, 2015;10(12):e0143854.
    Species: Human
    Sample Types: Tissue Homogenates
    Applications: Bioassay
  6. Tool compounds robustly increase turnover of an artificial substrate by glucocerebrosidase in human brain lysates.
    Authors: Berger, Zdenek, Perkins, Sarah, Ambroise, Claude, Oborski, Christin, Calabrese, Matthew, Noell, Stephen, Riddell, David, Hirst, Warren D
    PLoS ONE, 2015;10(3):e0119141.
    Species: Human
    Sample Types: Tissue Homogenates
    Applications: Enzyme Assay

FAQs

  1. Why is DTT (dithiothreitol) added to the assay buffer in the assay described for Recombinant Human Glucosylceramidase/GBA Protein, CF, Catalog # 7410-GHB?

    • The 5 mM DTT is added to the assay buffer in the assay protocol for catalog #  7410-GHB to prevent interchain disulfide bond formation that can cause aggregation of the protein and loss of enzyme activity over time. 

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