Bio-techne Corporation

Massachusetts Institute of Technology

Research & Diagnostics Systems, Inc.

Zhengliang L Wu

Robert D Rosenberg

Xinyi Huang

Cheryl M Ethen

A method for assaying endoglycosidase activity includes providing a proteoglycan having a glycosaminoglycan chain with a non-reducing end; treating the proteoglycan with a glycosyltransferase to incorporate a carbohydrate into the non-reducing end of the glycosaminoglycan chain, wherein the carbohydrate includes a click chemistry moiety; adding a label to the proteoglycan, wherein the label includes a click chemistry moiety that reacts to the click chemistry moiety of the carbohydrate such that the label attaches to the carbohydrate to form a labeled proteoglycan; immobilizing the labeled proteoglycan on a multi-well plate, wherein the multi-well plate includes a specific anti-proteoglycan antibody for binding the labeled proteoglycan; treating the labeled proteoglycan with an endoglycosidase specific to the glycosaminoglycan chain; and detecting the labeled proteoglycan.

Methods and kits for assaying endoglycosidase activity

Methods and kits for detecting the presence of GlcNAc modification of a peptide or protein. The steps of detection may include combining the peptide or protein with PAPS and a sulfotransferase to produce a reaction product includingS labeled GlcNAc modified protein or peptide, removing background labeling with a glycosidase, separating theS labeled GlcNAc modified protein or peptide from PAPS, free S-35 sulfate and labeled oligosaccharides if present, and detecting isotopic emissions from theS labeled GlcNAc modified peptide or protein. The detection may further include, prior to detection, the step of combining the peptide or protein with a GlcNAc transferase and UDP-GlcNAc under appropriate conditions for the GlcNAc transferase to transfer a GlcNAc to the peptide or protein.

Methods and kits for detection of O-GlcNAc and N-GlcNAc modification of peptides and proteins

A kit for detecting or measuring glycosyltransferase activity including a first reagent comprising a phosphatase and a second reagent comprising a free phosphate detector, and method of detecting and measuring glycosyltransferase activity. A sugar donor, an acceptor substrate, a glycosyltransferase enzyme and a phosphatase are combined and the amount of free phosphate present in the product is measured and used to calculate the activity of the glycosyltransferase enzyme.

Phosphatase coupled glycosyltransferase assay

A method of detecting sulfotransferase activity including conducting a first reaction comprising, measuring free phosphate produced by the first reaction, and comparing the measured free phosphate to a free phosphate standard curve or equation or to a free phosphate level obtained in a separate reaction. The first reaction includes combining the sulfotransferase, Golgi-resident PAP-phosphatase (gPAPP), a 5'-nucleotidase, a substrate of the sulfotransferase, and 3&#x2032;-phosphoadenosine-5&#x2032;-phosphosulfate (PAPS) under conditions to produce 3&#x2032;-phosphoadenosine-5&#x2032;-phosphate (PAP).

Sulfotransferase assay

Assays and methods for detecting and/or quantifying activity of a test sulfotransferase comprising by releasing a free phosphate from the PAP produced by the sulfotransferase reaction and detecting the free phosphate. The assay can includes gPAPP, a free phosphate detector and an optional control sulfotransferase. The method includes combining a test sulfotransferase with the substrate of the sulfotransferase, PAPS and gPAPP and detecting the free phosphate. The level of free phosphate directly correlates to the activity of the sulfotransferase. The assay and methods can be used to screen agents for their effect upon sulfotransferase activity.

The invention relates to methods for detecting and characterizing enzymatic modifications of oligosaccharides, such as heparan sulfate, and their interaction with binding partners, such as proteins, using an oligosaccharide-binding partner binding assay, such as a gel mobility shift assay. The instant invention relates to a rapid, convenient, sensitive and inexpensive method for identifying or studying oligosaccharide-binding partner interactions, identifying and characterizing structural features on oligosaccharides, identifying and characterizing binding partners, identifying agents capable of interfering with, enhancing, or facilitating the binding of an oligosaccharide to its binding partner, diagnosing conditions associated with altered oligosaccharide-binding partner binding, and generating oligosaccharide libraries and kits therefor.

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Edina, MN, United States of America

Zhengliang L Wu