Recombinant Human MAN1A1 His-tag Protein, CF Summary
Product Specifications
A distinct band is observed in the rhMAN1A1 digested sample on SDS-PAGE gel, as measured under the described conditions.
Pro63-Glu653, 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.
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.
|
Assay Procedure
- 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:
- Dilute rhMAN1A1 to 20 µg/mL in Digestion buffer.
- Dilute Man-9 to 20 µg/mL in Digestion Buffer.
- 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.
- Incubate at 37 °C for 2 hours.
Labeling:
- Dilute rhMGAT1 to 100 µg/mL in Labeling Buffer.
- Dilute UDP-GlcNAc to 1 mM in Labeling Buffer.
- Dilute rhFUT8 to 100 µg/mL in Labeling Buffer.
- Dilute GDP-Cy5-Fucose to 0.05 mM in Labeling Buffer.
- 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.
- Incubate at 37 °C for 60 minutes.
- Add 6 µL of Reducing SDS-PAGE gel loading buffer to each reaction.
- Load 12 µL of each reaction onto a 15% SDS-PAGE gel and perform electrophoresis.
- Analyze gel on a fluorescent imager.
- 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
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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.
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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.
Reconstitution Calculator
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).
- Oliveira-Ferrer, L. et al. (2014) Br. J. Cancer 110:753.
- Moremen K.W. et al. (2012) Nat. Rev. Mol. Cell Biol. 13:448.
- Moremen, K.W. and Nairn, A.V. (2014) Handbook of Glycosyltransferases and Related Genes p1297.
- Liu, T. et al. (2014) PLoS One 9:e107941.
- Milde-Langosch, K. et al. (2014) Breast Cancer Res. Treat. 145:295.
- Karen Legler, et al. (2018) Br. J. Cancer 118:847.
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