New England Biolabs, P0777S, Endo-β-Galactosidase

Related Categories Endoglycosidases Applications Glycoprotein Analysis Specification Unit Definition The enzyme activity is determined by its ability to cleave over 95% of 2 nmol Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc-7-amino-4-methyl-coumarin (AMC) using less than 10 ng of enzyme within 1 hour at 37°C, in a total reaction volume of 20 μl. Reaction Conditions 1X GlycoBuffer 3 Incubate at 37°C 1X GlycoBuffer 3 50 mM sodium acetate (pH 6 @ 25°C) Storage Buffer 50 mM sodium acetate 50 mM NaCl pH 5.5 @ 25°C Heat Inactivation 55°C for 10 minutes Molecular Weight Apparent: 40.8 kDa Unit Assay Conditions Two fold dilutions of Endo-β-Galactosidase are incubated with 2 nmol AMC labeled substrate in 1X GlycoBuffer 3 in a 20 μl reaction. The reaction mix is incubated at 37°C for 1 hour. Separation of reaction products are visualized via thin layer chromatography. FAQ Q: Do detergents inhibit Endo- β-Galactosidase (NEB #P0777)? A: Endo-β-Galactosidase activity can be inhibited by ionic detergents, such as SDS, at moderate concentrations (0.5–1%). To counteract this inhibition, it is essential to include NP-40 at a final concentration of 1% in your reaction mixture. In contrast, non-ionic detergents such as Tween 20, Triton X-100, and NP-40 can stabilize Endo-β-Galactosidase and enhance its specific activity. Q: How much Endo-β-Galactosidase (NEB #P0777) should be used in a reaction? A: The required amount of Endo-β-Galactosidase depends on the specific substrate. As an initial guideline, use 1–2 μl of enzyme for 10-20 μg of glycoprotein in a 10 μl reaction for one hour at 37°C. In our studies, with a sugar-conjugated protein, we observed that the enzyme functions effectively across a broad range of protein-to-enzyme molar ratios, from 2:1 to 150:1, which translates to a roughly 50- to 3500-fold sugar moiety-to-enzyme ratio under optimal conditions. We recommend beginning with a 10:1 to 20:1 ratio and conducting a titration experiment to optimize conditions for your specific substrate. Q: Can I use a different buffer than GlycoBuffer 3 supplied with Endo-β-Galactosidase (NEB #P0777)? A: Yes, you can use a different buffer, as our Endo-β-Galactosidase (EBG) is active across a broad pH range (pH 3.5–9). However, optimal cleavage occurs near pH 6.0. At pH 8.0, activity decreases to less than 50%, and is even further reduced at pH 8.5 after 1 hour at 37°C in our standard assay. To improve cleavage under non-optimal pH conditions, extend the incubation time, increase the enzyme amount, or both. Additionally, buffer composition and salt concentration can affect efficiency. For instance, in our experiments, a 50 mM sodium phosphate buffer (pH 7.4) performed better than 1x PBS at the same pH. Q: What if I have incomplete digestion when using Endo-β-Galactosidase (NEB #P0777)? A: The amount of enzyme required for complete digestion depends on the substrate's structure, purity, and quantity. Modifications such as branching or fucosylation can reduce or inhibit enzymatic cleavage, and oligosaccharides in the neolacto-group may be cleaved at significantly reduced rates. To address incomplete digestion, you can extend the incubation time, increase the enzyme concentration, or apply a combination of both. However, incubation beyond 8 hours at 37°C is unlikely to result in significant improvement. Q: What is the difference between Endo-β-Galactosidase, β1-4 Galactosidase, β1-4 Galactosidase S and β1-3,4 Galactosidase? A: Endo-β-Galactosidase (NEB #P0777S) is an endoglycosidase, whereas β1-4 Galactosidase (NEB #P0730S), β1-4 Galactosidase S (NEB #P0745S), and β1-3,4 Galactosidase (NEB #P0746S) are exoglycosidases. These enzymes are derived from different organisms and target distinct substrates. Endo-β-Galactosidase is cloned from Flavobacterium keratolyticus, β1-4 Galactosidase from Bacteroides fragilis, β1-4 Galactosidase S from Streptococcus pneumoniae, and β1-3,4 Galactosidase (BTG) from bovine testis. Endo-β-Galactosidase cleaves internal β(1-4)-galactose linkages within poly-N-acetyllactosamine structures [Galβ(1-4)-↓-GlcNAcβ(1-3)]. In contrast, β1-4 Galactosidase and β1-4 Galactosidase S cleave external galactose residues via β(1-4) linkages, while β1-3,4 Galactosidase (BTG) cleaves external galactose residues linked via both β(1-3) and β(1-4) linkages.