New England Biolabs, P0770S, p0770-idez-protease-igg-specific

IdeZ Protease (IgG-specific) is a recombinant antibody specific protease that recognizes human, sheep, monkey, rabbit, and some murine IgG subclasses, cleaving specifically at a single recognition site below the hinge region, to yield a homogenous pool of F(ab´)2 and Fc fragments. IdeZ Protease more effectively cleaves murine IgG2a than IdeS. Related Categories Proteases,, Proteome Analysis Applications Biotherapeutics and Antibody Analysis,, Proteomics Specification Unit Definition One unit is defined as the amount of enzyme required to cleave > 95% of 1 µg of human IgG, in 15 minutes at 37°C in a total reaction volume of 10 μl. Reaction Conditions 1X GlycoBuffer 2 Incubate at 37°C 1X GlycoBuffer 2 50 mM Sodium Phosphate (pH 7.5 @ 25°C) Storage Buffer 20 mM Tris-HCl 50 mM NaCl 1 mM EDTA pH 7.5 @ 25°C Heat Inactivation 65°C for 10 minutes Molecular Weight Theoretical: 35578 kDa Unit Assay Conditions Two fold dilutions of IdeZ Protease (IgG-specific) are incubated with 1 μg of human IgG and 1X GlycoBuffer 2 in a 10 μl reaction. The reaction mix is incubated for 15 minutes at 37°C. Separation of reaction products are visualized by SDS-PAGE. Storage Notes Avoid repeated freeze/thaw cycles. FAQ Q: What is the advantage of IdeZ over IdeS? A: IdeZ and IdeS have the same specificity; both cleave at the exact same single site below the hinge region of IgG. However, IdeZ is more versatile as it cleaves mouse IgG2a more efficiently than IdeS. Q: Is IdeZ Protease active in Phosphate Buffered Saline (PBS)? A: Yes, IdeZ Protease is active in PBS. IdeZ Protease also works in a range of buffers from pH 6.0 to pH 9.0. There is a significant decrease in activity at pH 5.5 and lower. The recommended buffer is 1X Glycobuffer 2 (50 mM Sodium Phosphate, pH 7.5). Q: Can protein A magnetic beads (NEB# S1425S) be used to create Fc and Fab fragment pools after cleavage with IdeZ Protease? A: Yes, Protein A magnetic beads (NEB# S1425S) can be used to create Fc and Fab fragment pools after cleavage with IdeZ Protease. The Fc fragment will bind to the beads and the Fab fragment will remain in the supernatant. Q: Can IdeZ Protease cleave IgGs other than human? A: IdeZ Protease is active on human IgG. It can also be used to prepare mouse IgG2a and IgG3 fragments with extended incubation time. IdeZ Protease will also cleave many Fc-fusion proteins as well as antibody drug conjugates (ADCs). IdeZ Protease is active on some monkey, rabbit, and sheep IgG. IdeZ Protease does not cleave goat, bovine, porcine, or rat IgG. It also will not cleave mouse IgG1 or IgG2b. Q: Can IdeZ Protease cleave Ig isotypes other than IgG? A: No, IdeZ Protease is only active on IgG. IdeZ is not active on IgA, IgD, IgE or IgM. Q: Can PNGase F be used together with IdeZ Protease under native conditions to deglycosylate the Fc portion of an antibody? A: Yes, PNGase F and IdeZ Protease can be used together in a single step to fragment the IgG and remove Fc glycans. Typical reaction conditions can be found in the simultaneous digestion of IgG with IdeZ Protease and PNGase F protocol. Q: What is the cleavage site for IdeZ Protease (IgG-specific)? A: Human IgG1, IgG3, IgG4: CPAPELLG / GPSVF.. Human IgG2: CPAPPVA / GPSVF Murine IgG2a: CPAPELLG / GPSVF