Recombinant Human Sulfamidase/SGSH Protein, CF Summary
Arg23-Leu502 with a C-terminal 6-His tag
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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.
|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 Buffer: 50 mM MES, pH 5.5
- Recombinant Human Sulfamidase/SGSH (rhSGSH) (Catalog # 8380-SU)
- Substrate: 4-Nitrocatechol Sulfate (PNCS) (Sigma, Catalog # N7251), 100 mM stock in deionized water
- Sodium Hydroxide (NaOH) (Sigma, Catalog # 221465), 2 M stock in deionized water
- 96-well Clear Plate (Catalog # DY990)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rhSGSH to 40 µg/mL in Assay Buffer.
- Dilute PNCS to 20 mM in Assay Buffer.
- Combine 150 µL of 40 µg/mL of rhSGSH and 150 µL of 20 mM of PNCS in microtubes. Include a Substrate Blank with 150 µL of Assay Buffer and 150 µL of 20 mM of PNCS.
- Incubate at 37 °C for 72 hours.
- Stop reactions and Substrate Blank by adding 300 µL of 0.2 M NaOH to each tube.
- Load 200µL from each stopped reaction and Substrate Blank vial into a plate.
- Read plate at 510 nm (absorbance) in endpoint mode.
- Calculate specific activity:
Specific Activity (pmol/min/µg) =
|Adjusted Abs* (OD) x Conversion Factor** (pmol/OD)|
|Incubation time (min) x amount of enzyme (µg)|
*Adjusted for Substrate Blank
**Derived using calibration standard 4-Nitrocatechol (PNC) (Sigma, Catalog # N15553).
Recombinant Human Sulfamidase/SGSH Protein, CF (Catalog # 8380-SU) hydrolyzes N-linked sulfate group from the non-reducing terminal glucosaminide residues of heparan sulfate.
2 μg/lane of Recombinant Human Sulfamidase/SGSH Protein (Catalog # 8380-SU) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 55-63 kDa.
Also known as N-sulfoglucosamine sulfohydrolase and heparan N-sulfatase, Sulfamidase/SGSH is an important member of the sulfatase family involved in the degradation of heparan sulfate (HS) (1). Different from the HS specific endosulfatases that remove sulfate from internal GlcNAc residues (2), SGSH removes sulfate group from the non-reducing end glucosamine residues on HS. The SGSH deficiency results in mucopolysaccharidosis type IIIA (MPS IIIA, Sanfilippo A syndrome), an autosomal recessive lysosomal storage disease characterized by neurological dysfunction but relatively mild somatic manifestations (3). Human SGSH shows 88.6% sequence identity with that of mouse sequence.
- DiezRoux, G. and Ballabio, A. et al. (2005) Annu. Rev. Genomics Hum. Genet. 6:355.
- Morimoto-Tomita, M. et al. (2002) J. Biol. Chem. 277:49175.
- Blanch, L. et al. (1997) Hum. Mol. Genet. 6:787.
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