Endo-β-N-acetylglucosaminidase (Endo A)

Instructions for Use
Endo A (Endoglycosidase A) is derived from Arthrobacter protophormiae and is expressed in E. coli. Featuring an N-terminal His-tag, it is a highly specific endoglycosidase capable of cleaving high-mannose and hybrid-type N-glycans-specifically those lacking core fucosylation-from glycoproteins. This product exhibits high enzymatic activity and excellent stability, making it suitable for use in antibody glycosylation modification and analysis. The buffer composition consists of 20 mM Tris-HCl, 200 mM NaCl (pH 7.5), and 10% glycerol.
Preparation Before Use
1. Prior to use, remove the reagent from storage, add 30-50 μL of sterile deionized water to dissolve it, and centrifuge at 10,000 rpm for 10 seconds to ensure that all reagent settles at the bottom of the tube.
2. Retrieve the 10× Glycoprotein Denaturing Buffer, 10× GlycoBuffer, and 10% NP-40 solution (stored at -20°C) and set them aside for use.
Note: Prepare reagents according to the specific requirements of your experiment; for instance, if performing a deglycosylation reaction under non-denaturing conditions, the 10× Denaturing Buffer and 10% NP-40 solution are not required.
Usage Instructions
1. Glycoprotein Deglycosylation under Denaturing Conditions
1) Dissolve 1-20 µg of glycoprotein in deionized water; add 1 µL of 10× Glycoprotein Denaturing Buffer; and bring the final volume to 10 µL using deionized water.
2) Incubate at 75°C for 10 min.
3) Add 2 µL of 10× GlycoBuffer and 2 µL of 10% NP-40; gently pipette to mix.
4) Add 1-4 µL of Endo A; add deionized water to bring the final volume to 20 µL; and gently pipette to mix.
5) Incubate at 37°C for 1-4 hours.
6) Proceed to SDS-PAGE or HPLC analysis.
2. Glycoprotein Deglycosylation under Non-denaturing Conditions
1) Take 10-100 µg of glycoprotein solution; add 2 µL of 10× GlycoBuffer and 2-5 µL of Endo A; add purified water to bring the total reaction volume to 20 µL; and mix gently.
2) React at 37°C for 4-24 hours.
Note: 1. When deglycosylating native glycoproteins, it is recommended to run a parallel enzymatic digestion experiment using an equal amount of denatured glycoprotein sample as a positive control. This helps determine the extent of the deglycosylation reaction under non-denaturing conditions.
2. When scaling up the reaction volume, the incubation time or enzyme amount should be adjusted according to actual requirements. Gentle shaking or mixing at a low speed can be applied to help increase the conversion efficiency.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.