Active Recombinant O-Glycoprotease, His tagged

• Cat.No.: O-Glycoprotease-13
• Product Overview: O-glycoprotease is expressed in E.coli. This enzyme contains a 6×His tag, which allows for convenient removal after the reaction.

Specification

• Source: E.coli
• Tag: His
• Description: It is a highly specific glycoprotease that can specifically recognize the serine or threonine residues of mucin-type O-linked glycans (whether sialylated or not) and cleave at their N-termini. It can generate O-glycopeptides suitable for mass spectrometry analysis.
• Purity: ≥ 95% by SDS-PAGE
• Advantages: 1. Wide adaptability: It is active against O-glycoproteins with or without sialic acid, and shows higher activity on desialylated O-glycoproteinsIt can effectively cleave glycoproteins without the need for neuraminidase treatment; 2. High purity: It is free from contamination by other proteases and has no other endo- and exo- glycosidase activities, with a purity of ≥ 95%; 3. High stability: Each batch of O-glycoprotease undergoes strict quality control to ensure batch-to-batch stability of the product; 4. Compatible with HPLC/MS: It does not contain glycerol, which helps to achieve optimal results in HPLC and mass spectrometry analyses; 5. Easy removal: It contains a 6×His-Tag and can be easily removed from the reaction system using nickel-affinity resin.
• Protein Content: ≥ 1 U/μL
• Unit definition: One unit is defined as the amount of the enzyme required to cleave more than 90% of 2 μM O-glycopeptide in a 20 μL reaction system containing 20 mM Tris-HCl, pH 8.0, at 37 centigrade for 2 hours.
• Usage: 1. Mix 10 μg of glycoprotein with a solution of 20 mM Tris-HCl (pH 8.0), 40 mM DTT, and 0.1% SDS to make the total volume 50 μL. 2. Heat at 95 centigrade for 10 minutes. 3. Cool to room temperature. 4. Add 3 μL of 500 mM iodoacetic acid solution. 5. Leave it at room temperature for 30 minutes. 6. Transfer the solution to an ultrafiltration centrifuge tube. 7. Centrifuge at 12, 000 × g for 4 minutes and discard the flow-through. 8. Add 400 μL of 20 mM Tris-HCl (pH 8.0) solution. 9. Centrifuge at 12, 000 × g for 4 minutes and discard the flow-through. Repeat this step by adding another 400 μL of 20 mM Tris-HCl (pH 8.0) solution. 10. Add 20 μL of 20 mM Tris-HCl (pH 8.0) solution. 11. Add 1 μL of O-Glycoprotease and mix gently. 12. Incubate at 37 centigrade for 2-18 hours. 13. Centrifuge at 12, 000 × g for 4 minutes and collect the supernatant.
• Applications: 1. Determination of O-glycosylation sites 2. Determination of O-glycan structures
• Note: 1. Try to avoid freeze-thaw cycles of this product after receipt; 2. Please wear lab coat and disposable gloves when using; 3. This product should not be used directly for clinical diagnosis and treatment.
• Stability: This product should be stored at -20 centigrade and can be stored for at least 12 months.
• Storage: Store at -20 centigrade.
• Storage Buffer: 20mM Tris-HCl, 100mM NaCl, pH 7.5
• Shipping: Dry Ice