glycoproteins Protocols

Category: Molecular Biology Protocols: Carbohydrate Protocols

Deglycosylation steps are different with every glycoprotein and must be determined empirically (Methods in Enzymology, 1994, 230: 44-57). A typical test reaction of human transferrin is described. 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 - [Read Deglycosylation of Glycoproteins Using Endoglycosidases]

Category: Transfection Protocols: Stable Transfection Protocols

CHO Lec3.2.8.1 cells have 4 independent mutations in the N and O glycosylation pathways. When cultured with alpha-glucosidase I inhibitor N-butyl-deoxynojirimycin, glycoproteins produced in CHO Lec3.2.8.1 cells are completely susceptible to Endo H digestion. Endo H cleaves chitobiose, leaving a single N-linked N-acetylglucosamine per site which is ideal for maintenance of protein solubility and special carb-protein interactions, such as between the first N-acetyl glucosamine residue and tryp. - [Read Establishment of Stable Transfectant of CHO Lec Cells Protocol]

Category: Chromatography Protocols: Affinity Chromatography Protocols

Two methods are provided for purifying glycoproteins using wheat-germ agglutinin or concanavalin A-Sepharose.  Because lectin-affinity matrices can bind a few milligrams of protein per milliliter of affinity matrix, only a small amount of affinity gel matrix is required.  The batchwise method is recommended when protein volume is large. - [Read Lectin-Agarose Affinity Chromatography Protocol]

Category: Immunology: Immunoprecipitation Protocols: Immunoprecipitation of Radiolabeled Cells Protocols

Cells incorporate 35S-methionine or cysteine during the protein synthesis. Thus it is essential to use Met,Cys-free medium and dialyzed FCS during the labeling. Short period of incubation with 35S-methionine or cysteine will result in radiolabeling (pulse), and additional incubation with excess concentration of unlabeled Met+Cys (chase) is needed for complex glycoproteins like integrins to get expressed as a maturated form. - [Read Metabolic Labeling & Immunoprecipitation Protocols]

Category: Chromatography Protocols: Gas Chromatography Protocols

Protocol presents a method that allows rapid determination of the monosaccharide composition of glycans, glycoproteins, and proteoglycans that contain (or are suspected to contain) phosphorylated sugars.  (For samples that do not contain phosphorylated sugars see Monosaccharide Analysis by Methanolysis.) - [Read Monosaccharide Analysis of Phosphorylated Sugars by Methanolysis and Diazomethane Methylation]

Category: Chromatography Protocols: Gas Chromatography Protocols

Protocol describes how to determine the monosaccharide composition of glycans, glycoproteins, or proteoglycans by hydrolyzing the sample to monosaccharides and converting them to alditols, then performing acetylation to make them volatile compounds and analysis by gas chromatography (GC) or gas chromatography coupled with mass spectrometry (GC-MS). - [Read Monosaccharide Composition Analysis: Alditol Acetates Protocol]