Recombinant Human MAN1A1 His-tag Protein, CF

Catalog # Availability Size / Price Qty
10665-GH-050
Recombinant Human MAN1A1 His-tag Protein Enzyme Activity Diagram.
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Recombinant Human MAN1A1 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
<1.0 EU per 1 μg of the protein by the LAL method.
Activity
Measured by its ability to remove alpha -mannose from the high mannose glycan Man-9.
A distinct band is observed in the rhMAN1A1 digested sample on SDS-PAGE gel, as measured under the described conditions.
Source
Chinese Hamster Ovary cell line, CHO-derived human MAN1A1 protein
Pro63-Glu653, with a C-terminal 6-His tag
Accession #
N-terminal Sequence
Analysis
Pro63
Predicted Molecular Mass
67.7 kDa
SDS-PAGE
63-65 kDa, under reducing conditions.

Product Datasheets

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10665-GH

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.

10665-GH

Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl and CaCl2.
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
  • Digestion Buffer: 50 mM MES, 15 mM CaCl2, 1 mg/mL BSA, pH 6.0
  • Labeling Buffer: 25 mM Tris, 150 mM NaCl, 10 mM MnCl2, pH 7.5
  • Recombinant Human MAN1A1 (rhMAN-1A1) (Catalog # 10665-GH)
  • Oligomannose-9 (Man-9) (Dextra Laboratories, Catalog # MC1131), 0.1 mg/mL stock in deionized water
  • Recombinant Human MGAT1 (rhMGAT1) (Catalog # 8334-GT)
  • UDP-GlcNAc (Sigma, Catalog # U4375), 50 mM stock in 50% ethanol, 50% deionized water
  • Recombinant Human FUT8 (rhFUT8) (Catalog # 5768-GT)
  • GDP-Cy5-Fucose  (Catalog # ES301)
  • 15% SDS-PAGE gel
  • Reducing SDS-PAGE gel loading buffer
  • Fluorescent imager

Digestion:

  1. Dilute rhMAN1A1 to 20 µg/mL in Digestion buffer.
  2. Dilute Man-9 to 20 µg/mL in Digestion Buffer.
  3. Combine 5 µL of 20 µg/mL Man-9, 5 µL of 20 µg/mL rhMAN1A1 and 10 µL of Digestion Buffer. Include a Control containing 5 µL of 20 µg/mL Man-9 and 15 µL of Digestion Buffer.
  4. Incubate at 37 °C for 2 hours.

Labeling:

  1. Dilute rhMGAT1 to 100 µg/mL in Labeling Buffer.
  2. Dilute UDP-GlcNAc to 1 mM in Labeling Buffer.
  3. Dilute rhFUT8 to 100 µg/mL in Labeling Buffer.
  4. Dilute GDP-Cy5-Fucose to 0.05 mM in Labeling Buffer.
  5. Transfer 10 µL of each digestion to a new tube and add 5 µL of 100 µg/mL rhMGAT1, 5 µL of 1 mM UDP-GlcNAc, 5 µL of 100 µg/mL rhFUT8 and 5 µL of 0.05 mM GDP-Cy5-Fucose.
  6. Incubate at 37 °C for 60 minutes.
  7. Add 6 µL of Reducing SDS-PAGE gel loading buffer to each reaction.
  8. Load 12 µL of each reaction onto a 15% SDS-PAGE gel and perform electrophoresis.
  9. Analyze gel on a fluorescent imager.

Per Reaction:
  •  rhMAN1A1: 0.05 µg
  •  Man-9: 0.05 µg
  •  rhMGAT1: 0.5 µg
  •  UDP-GlcNAc: 5 nmol
  •  rhFUT8: 0.5 µg
  •  GDP-Cy5-Fucose: 0.25 nmol

Scientific Data

Enzyme Activity View Larger

MAN1A1 is involved in the removal of 4 distinct alpha -1,2-linked mannose residues from Man9GlcNAc2 to produce Man5GlcNAc2, an essential step of N-glycan maturation.

SDS-PAGE View Larger

1 μg/lane of Recombinant Human MAN1A1 His-tag (Catalog # 10665-GT) was resolved with SDS-PAGE under reducing (R) conditions and visualized by silver staining, showing a band at 63-65 kDa.

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Background: MAN1A1

N-glycan maturation in Golgi apparatus starts with high-mannose glycan Man-9 that is capped with four 1,2-alpha -linked mannose residues at its non-reducing ends. During the process, these mannose residues are removed to generate Man-5 oligomannose glycan, a precursor for complex and hybrid N-glycans (1). Failure of removing these mannose residues will result in the display of high-mannose glycans on cell surface and extracellular matrix. Increased levels of high-mannose glycans on cell surface are usually associated with disease progress such as tumorigenesis and viral infection (2). The removal of 1,2-alpha -linked mannose residues are catalyzed by 4 alpha -mannosidases, including MAN1A1, MAN1B1, MAN1A2 and MAN1C1, that have overlapping substrate specificity and slight differences in enzyme activity (3). MAN1A1 is also a tumor-suppressor (4) and low levels of expression of MAN1A1 correlate with poor prognosis in breast cancer patients (5, 6).

References
  1. Oliveira-Ferrer, L. et al. (2014) Br. J. Cancer 110:753.
  2. Moremen K.W. et al. (2012) Nat. Rev. Mol. Cell Biol. 13:448.
  3. Moremen, K.W. and Nairn, A.V. (2014) Handbook of Glycosyltransferases and Related Genes p1297.
  4. Liu, T. et al. (2014) PLoS One 9:e107941.
  5. Milde-Langosch, K. et al. (2014) Breast Cancer Res. Treat. 145:295.
  6. Karen Legler, et al. (2018) Br. J. Cancer 118:847.
Long Name
Mannosidase alpha, Class 1A, Member 1
Entrez Gene IDs
4121 (Human); 17155 (Mouse)
Alternate Names
HUMM3; HUMM9; MAN1A1; MAN9

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