Protocol 1

A Method for Micro-Scale Isolation and Purification of Gangliosides

Stephan Ladisch~Director, Center for Cancer and Transplant Biology, Children's National Medical Center, Washington, D.C. 22010

This solvent partition method permits the isolation of gangliosides from small samples and from samples in which the ganglioside concentration is low, especially relative to the concentration of potentially contaminating proteins and other large molecular weight species.

Protocol 2

Immunohistochemistry using Anti-Ganglioside Antibodies

Tadashi Tai~Head, Department of Tumor Immunology, The Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan

An indirect method using immuno- fluorescence coupled to second antibody is a relatively simple, rapid, and easy immuno-histochemical procedure. It is best suited for the study of membrane antigens in addition to intra- and extracellular antigens, and may be applied to frozen tissue sections, to cells in suspension, and to cells attached to glass slides or coverslips.

Protocol 3

Deglycosylation of Glycoproteins Using Endoglycosidases

T.H. Plummer, Jr. and A.L. Tarentino, Department of Biochemistry, New York State Department of Health, Wadsworth Center for Laboratories and Research, Albany, New York 12201

Parameters of deglycosylation vary with every glycoprotein and must be determined empirically (Methods in Enzymology, 1994, 230: 44-57). Human transferrin is an example of a glycoprotein containing two asparagine-linked -fucosyl-biantennary chains, one of which is only released slowly by endoglycosidases even after denaturation in SDS. A typical test reaction on human transferrin is described.

Protocol 4

A Sensitive ELISA-Based Assay for Glycosyltransferases

Bruce A. Macher~Professor of Chemistry and Biochemistry, San Francisco State University, San Francisco, CA 94132

This procedure allows the detection and quantification of glycosyltransferase activity using an ELISA-based procedure and carbohydrate-specific monoclonal antibodies. The procedure avoids the use of radiolabeled substrates, and provides a method for the detection of a specific product.

Protocol 5

Immunostaining Thin Layer Chromatograms Of Glycolipids

John L. Magnani~GlycoTech Corporation, Rockville, Maryland 20850

Immunostaining thin layer chromatograms allows for very sensitive detection of functionally active carbohydrate ligands of protein receptors. These carbohydrate structures are detected in glycolipids from complex mixtures of molecules extracted from the relevant target tissue. Some protein receptors that may be analyzed are: antibodies, chimeric Ig proteins, lectins, selectins, toxins, and other carbohydrate binding proteins.

Protocol 6

Analysis of Oligosaccharide Ligands by High Performance Liquid Affinity Chromatography

WeiTong Wang~GlycoTech Corporation, Rockville, Maryland 20850

High performance liquid affinity chromatography (HPLAC) is a useful technique for investigation of the interaction between a carbohydrate binding protein and its ligands. One of the major applications of HPLAC is to screen and separate natural ligands from complex biological mixtures such as detecting, purifying and determining the binding constant of a carbohydrate ligand from bodily fluids. HPLAC is also useful in the rational design and synthesis of new carbohydrate drugs by providing a fast,sensitive, and simple method for the determination of KD values.

Protocol 7

Dynamic Flow Assay in a Parallel Plate Flow Chamber

John T. Patton~GlycoTech Corporation, Rockville, Maryland 20850

Flow assays allow visualization of cell adhesion under well-defined wall shear stress. The visualization of the different events of cell adhesion can be quantified by selective image acquisition and subsequent image processing. Flow assays are uniquely suited to the investigation of adhesive events which occur very rapidly in a time scale shorter than that of most static adhesion assays. In addition, events subsequent to the initial events can be studied such as cell stabilization and spreading giving some insight into the kenetics of particular cell-cell or cell-substrate adhesive behavior.

Protocol 8

Measurement of Cell Adhesion Under Static Conditions

John L. Magnani~GlycoTech Corporation, Rockville, Maryland 20850

Many different molecules have been described to promote cell adhesion including several cell surface carbohydrate-binding proteins. Measuring cell adhesion in the convenient 96-well microtiter plate format is difficult due to the shear forces generated by washing the wells. This protocol introduces the use of a liquid-filled wash chamber that separates unbound cells by gravity thereby eliminating uncontrolled shear forces and passage of adherent cells through a liquid/air interface. This protocol is also useful for assaying molecules that promote or inhibit cell adhesion.

Protocol 9

Carbohydrate-Specific Adhesion of Intact Cells to Resolved Glycolipids on TLC Plates

Ronald L. Schnaar~Professor, Johns Hopkins University Medical School, Baltimore, Maryland 21205

This procedure allows detection of specific cell adhesion to glycolipids resolved on TLC plates.


Copyright 1996-99 by GlycoTech Corporation. All rights reserved.