Recombinant S. plicatus Endo H Protein, CF Summary
37 °C.. Use of Recombinant S. plicatus Endo-beta -N-acetylglucosaminidase H/Endo H in the deglycoslyation of other substrates may require alternative conditions for optimal performance.
Val47-Pro313 with an N-terminal Met and 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, NaCl and EDTA.|
|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 Sodium Phosphate, pH 5.5
- Recombinant S. plicatus Endo-beta -N-acetylglucosaminidase H/Endo H (rS. plicatus Endo H) (Catalog # 5224-GHB)
- RNase B (Sigma, Catalog # R1153), 1 mg/mL stock in 10 mM Sodium Phosphate, pH 5.5
- 4-20% SDS-PAGE gel
- Reducing SDS-PAGE gel loading buffer
- Silver Staining reagents
- Dilute Endo H to 2.5, 1.25, 0.625. 0.313, 0.156, 0.0781, and 0.0391 μg/mL in Assay Buffer.
- Dilute RNase B to 100 μg/mL in Assay Buffer.
- Combine 25 μL of Endo H at each dilution with 25 μL of 100 μg/mL RNase B. Include a control containing 25 μL Assay Buffer and 25 μL of 100 μg/mL RNase B.
- Incubate the reaction and control at 37 °C for 30 minutes.
- Combine 30 μL of each reaction with 30 μL of reducing SDS-PAGE gel loading buffer. Heat samples at 100 °C for 3 to 5 minutes before loading to gel.
- Load 40 μL of sample per lane (1 μg of RNase B per lane) on a 15% gel.
- Stain gel with silver stain.
- Determine the 50% deglycosylation concentration (DC50) for Endo H by plotting % of RNase B deglycosylated vs. Endo H concentration with 4-PL fitting.
- rS. plicatus Endo H: 25, 12.5, 6.25, 3.13, 1.56, 0.781, and 0.391 ng
- RNase B: 1 µg
Background: Endo-beta-N-acetylglucosaminidase H/Endo H
N-glycans are commonly found on various glycoproteins. While peptide N-glycosidase from Flavobacterium meningosepticum (PNGase F) is widely used to release virtually all types of N-glycans under denaturing conditions, some bacterial endo-beta -N-acetylglucosaminidases, including Endo H, can be used under native conditions to specifically release particular types of N-glycans (1, 2). Because these glycosidases hydrolyze the chitobiose core of an N-glycan on a glycoprotein, the released glycan product will contain one GlcNAc residue at its reducing end with the other GlcNAc residue remaining attached to the glycoprotein. Endo H specifically releases oligomannose and hybrid but not complex type N-glycans from glycoproteins (3-5). It also remains active on core fucosylated N-glycans but not sulfated glycans (5).
- Maley, F. et al. (1989) Anal. Biochem. 180:195.
- Tarentino, A.L. et al. (1985) Biochemistry 24:4665.
- Hsieh, P. et al. (1983) J. Biol. Chem. 258:2555.
- Tarentino, A.L. et al. (1992) J. Biol. Chem. 267:3868.
- Trimble, R.B. and Tarentino, A.L. (1991) J. Biol. Chem. 266:1646.
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