Recombinant S. pyogenes Endo S2 His-tag Protein, CF SummaryLearn more about Fluorescent Glycan Labeling and Detection
>50% of Cy5-Labeled Glycan G2 (0.2 pmol) is digested by 0.2 μg of rSp. Endo-S2, as measured under the described conditions.
Glu37-Asp843, with a N-terminal Met & 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 6.0
- Recombinant Sp. Endo-S2 (rSpEndo-S2) (Catalog # 10976-GH)
- Cy5-Labeled Glycan G2 (Cy5-G2) (Catalog # GL302)
- 15% SDS-PAGE gel and SDS-PAGE Reagents
- Reducing SDS-PAGE Sample Buffer
- Fluorescent imager
- Dilute rSpEndo-S2 to 20 ng/µL in Assay Buffer.
- Dilute Cy5-G2 to 0.02 µM in Assay Buffer.
- Prepare reaction by combine 10 µL of diluted rSpEndo-S2 and 10 µL of diluted Cy5-G2.
- Prepare a negative control by combining 10 µL of diluted Cy5-G2 and 10 µL of Assay Buffer.
- Incubate reaction(s) and negative control at 37 °C for 60 minutes.
- Add 7 µL of reducing SDS-PAGE sample buffer to each reaction and negative control.
- Load 13.5 µL of each sample and control per well on a 15% SDS-PAGE gel and run SDS-PAGE until the dye front has migrated more than two thirds of the way down the gel.
- Acquire gel image with a fluorescent imager.
- Analyze the amount (%) of Cy5-G2 digested by rSpEndo-S2 in each reaction lane.
- rSpEndo-S2: 0.20 µg
- Cy5-G2: 0.2 pmol
Endo S2 recognizes the conserved N-glycans on the Fc region of IgG by hydrolyzing the chitobiose core (at the beta -1,4 linkage between the two N-acetylglucosamines) of the N-glycans. Endo S2 leaves one GlcNAc residue attached the the asparagine of the peptide backbone. R1 and R2 can be oligosaccharide extensions containing Gal, GlcNAc, and Sialic Acid. R3 can be unmodified or Core-6 Fucose.
1 μg/lane of rSp. Endo-S2 (Catalog # 10976-GH) was resolved with SDS-PAGE under reducing (R) conditions and visualized by silver staining, showing a band at 92 kDa.
Lane 1 contained substrate Cy5-labeled G2 GL302. In the presence of Endo S2, the glycan was digested to products and the smaller product Fuc-alpha,6-GlcNAc is observed.
Background: Endo-beta-N-acetylglucosaminidase S2/Endo S2
Streptococcus pyogenes is a leading Gram-positive bacterial pathogen that can abolish the effector functions of human immunoglobulin G (IgG) through deglycosylation (1). Upon infection, the pathogen secret two endoglycosidases, Endo S and Endo S2, that specifically deglycosylate the conserved N-glycans on the Fc region of IgG by hydrolyzing the chitobiose core (at the beta -1,4 linkage between the two N-acetylglucosamines) of the N-glycans (2, 3). Endo S and S2 cleavage leave one GlcNAc residue remaining attached to the asparagine residue on the peptide backbone. The enzymes are highly specifc to native IgG molecules (3), suggesting that the local conformation of IgG is required for the enzymatic recognition. In comparison, PNGase F from Flavobacterium meningosepticum completely removes glycans from glycoproteins and is more active on denatured glycoproteins. Cleavage of the glycans from IgG antibodies by Endo S and S2 result in conformation change of the antibodies thereby dramatically diminish the binding affinity to their receptors (4) and abolish their opsonizing functions (5, 6). By using fluorophore labeled N-Glycans as substrates, we found that Endo S2 has similar activity to Endo S towards non-galactosylated IgG glycan species including oligomannose and hybrid glycans but shows much high activity on galactosylated and sialylated IgG glycan species. We also found that both enzymes are highly active on core-6 fucosylated IgG glycans but with no activities on those with bisecting GlcNAc.
- Nizet, V. (2007) J. Allergy Clin. Immunol. 120:13.
- Collin, M. and Olsen, A. (2001) EMBO J. 20:3046.
- Sjogren, J. et al. (2013) Biochem. J. 455:107.
- Allhorn, M. et al. (2008) PLoS ONE. 3:e1413.
- Collin, M. et al. (2002) Infect Immun. 70:6646.
- Sjögren, J. et al. (2011) BMC Microbiol. 11:120.
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