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Rapid Quantitation of IgG after Digestion at Elevated Temperature with a Novel Trypsin Reagent

Part # PS298

Abstract

Michael M. Rosenblatt1, Sergei Saveliev1, Daniel Spellman2, Kevin Bateman2, and Marjeta Urh1
1Promega Corporation, 2800 Woods Hollow Rd, Madison, WI, 53711
2Merck & Co., Incorporated , West Point PA, 19486

As increasing numbers of protein-based therapeutics enter development pipelines, more efficient protocols are required for structural characterization and quantitation. Proteolysis of these proteins into peptides represents a bottleneck and often requires optimization of numerous steps including reduction, alkylation and digestion time. We have developed a new Trypsin reagent, Rapid Digestion--Trypsin and Rapid Digestion--Trypsin with Lys C, that streamlines the entire sample preparation process to less than 1 hour. With this new process, proteolysis is performed at 70°C which facilitates both denaturation and rapid digestion. This protocol is robust, amenable to multiple analytes including pure proteins and complex mixtures, and is compatible with digestion under native, reduced and/or denaturing conditions.

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