Recombinant Human Sialate O-acetylesterase/SIAE His-tag, CF Summary
Product Specifications
Ile24-Lys523, 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.
11497-SI
| Formulation | Supplied as a 0.2 μm filtered solution in Tris and NaCl. |
| 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 Tris, pH 8.0
- Recombinant Human SIAE His tag (rhSIAE) (Catalog # 11497-SI)
- Substrate: 4-Nitrophenyl Acetate, 100 mM stock in acetone
- Clear 96 Well Plate
- Plate Reader with Absorbance Read Capability
- Dilute rhSIAE to 1.2 μg/mL in Assay Buffer.
- Dilute Substrate to 8 mM in Assay Buffer.
- Load into a plate 50 μL of 1.2 μg/mL rhSIAE, and start the reaction by adding 50 μL of 8 mM Substrate. For Substrate Blank, load 50 μL of Assay Buffer and 50 μL of 8 mM Substrate.
- Read plate at 410 nm in kinetic mode for 5 minutes.
- Calculate specific activity:
Specific Activity (pmol/min/µg) = | Adjusted Vmax* (OD/min) x well volume (L) x 1012 pmol/mol |
| ext. coeff** (M-1cm-1) x path corr.*** (cm) x amount of enzyme (µg) |
- rhSIAE: 0.06 μg
- Substrate: 4 mM
Scientific Data
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Recombinant Human SIAE His-tag Protein (Catalog # 11497-SI) catalyzes the removal of O-acetyl ester groups from position 9 of Sialic Acid.
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Recombinant Human SIAE His-tag Protein (Catalog # 11497-SI) is measured by its ability to hydrolyze p-nitrophenylacetate.
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2 μg/lane of Recombinant Human SIAE His-tag Protein (Catalog # 11497-SI) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 63-69 & 88-97 kDa, under reducing conditions.
Background: SIAE
Recombinant human Sialate O-acetylesterase (SIAE) is a glycosylated lysosomal protein that has two transcript isoforms: the cytosolic sialic acid esterase (Cse) that lacks 35 amino acids at the N-terminus and the lysosomal/membrane-associated sialic acid esterase (Lse) (1, 2). The Lse form is the predominantly expressed form responsible for the acetyl esterase activity on NeuAc in most tissues (2) and represents the recombinant product form. SIAE contains a signal sequence, a variable N-terminus, and a core protein structure that is unique within the SGNH hydrolase family (2) and contains a shallow active site pocket that accommodates binding of various complex substrates with acetylated sialic acid. SIAE Lse form catalyzes the removal of O-acetyl ester groups from position 9 of Sialic acid and is highly expressed in the testis, colon, and prostrate (3). SIAE deficiency has been shown to impact B cell development, signaling, and immunological tolerance through modulation of Siglec binding (4). Many SIAE variants in humans that result in defective activity or improper secretion have been identified in patients with autoimmune diseases including Crohn's disease, Sjogren's syndrome, lupus, rheumatoid arthritis, and type I diabetes (5). In addition, SIAE modulation of Siglec binding and sialic acid levels may play a key role in the immune evasion pathway associated with cancer (6). SIAE has also been implicated to play an important role in immune modulation during pregnancy (7).
- Higa, H.H. et. al. (1987) Biochem. Biophys. Res. Commun. 144:1099.
- Orizio, F. et. al. (2015) Glycobiology 9:992.
- Zhu, H. et. al. (2004) J. Biomed. Biotechnol. 3:130.
- Cariappa, A. et. al. (2009) J. Exp. Med. 206:125.
- Surolia, I. et. al. (2010) Nature 466:243.
- Grabenstein, S. et. al. (2021) Glycobiology 31:1279.
- Erickson, J. et. al. (2022) Nature 606:769.
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