A re-evaluation of the surface complexity of the intact erythrocyte

Surface proteins and glycoproteins of intact human red blood cells were labelled with ¹²⁵I by the lactoperoxidase method. The radioactive proteins were then separated in each of the Fairbanks and Laemmli one-dimensional polyacrylamide gel electrophoresis systems. The radioactive polypeptides had different mobilities in the two systems, largely due to the anomalous migration of glycoproteins in polyacrylamide gels. A two-dimensional system was therefore developed using the Fairbanks and Laemmli buffer systems to exploit these anomalies. This procedure clearly resolved radioactive glycoproteins and proteins and enabled the identification of many more surface components than had previously proved possible.     

Concanavalin A-horseradish peroxidase bridge staining of alpha-1 glycoproteins separated by isoelectric focusing on polyacrylamide gel.

The Coomassie Blue protein stain and the periodic acid-Schiff stain for glycoproteins are compared to a new method of staining glycoproteins resolved electrophoretically. The method utilizes a Concanavalin A-horseradish peroxidase sequence to visualize selectively glycoproteins with terminal or internal mannose or terminal N-acetylglucosamine. The method applied to characterization of M and Z allele products of alpha-l-antitrypsins separated by isoelectric focusing of polyacrylamide gels slabs have revealed differences in carbohydrate content of various components that were previously undetected.     

Direct tissue isoelectric focusing on ultrathin polyacrylamide gels. Applications in enzyme, lectin and immunohistochemistry

Summary Application of cryostal sections directly onto ultrathin polyacrylamide gels and subsequent isoelectric focusing allows elution of proteins, glycoproteins and peptides out of the sections into the gels. The eluted compounds reveal clearly delineated band patterns in the polyacrylamide gels. The advantage of this method is that enzyme histochemical reactions can be directly performed in the gel and in the electroeluted tissue sections. Therefore, this method is suitable for specifying, in more detail, histochemical enzyme reactions and for detecting multiple forms of enzymes even from a single tissue section. Furthermore, the transfer of proteins, glycoproteins and peptides from the gel onto nitrocellulose by a modified Western blot procedure offers the possibility of checking findings obtained by lectin histochemistry and immunohistochemistry.     

The visualization of human erythrocyte membrane proteins and glycoproteins in SDS polyacrylamide gels employing a single staining procedure.

Human erythrocyte membrane proteins and glycoproteins were visualized after separation by sodium dodecyl sulfate polyacrylamide gels into molecular weight classes using a single staining procedure with a cationic carbocyanine dye (“Stains-all”). The sialoglycoproteins stained blue; the proteins, red; and the lipids, yellow-orange. This method is useful in detecting simultaneously the position of proteins and sialoglycoproteins in the commonly used SDS polyacrylamide gel electrophoresis.     

Enhanced detection of glycoproteins in polyacrylamide gels

A highly sensitive and simple method to enhance detection of glycoproteins resolved by either one- or two-dimensional polyacrylamide gel electrophoresis is described. The method is a modification of the procedure described by D. Fargeaud et al. (D. Fargeaud, J. C. Benoit, F. Kato, and G. Chappuis (1984) Arch. Virol. 80, 69-82) that uses concanavalin A conjugated with fluorescein isothyocyanate to detect the carbohydrate moiety of glycoproteins. Briefly, the electrophoresed gel is exposed to the fluorescent lectin, thoroughly washed, and sequentially transferred to 50% methanol in deionized water and to absolute methanol. The result is an abrupt dehydration of the gel which turns evenly white and stiff. At least a twofold enhancement of fluorescence is obtained as detected by exposing the treated gel to an appropriate uv source. The sensitivity of the procedure allows us to detect purified immunoglobulin molecules by their carbohydrate content in the range of 0.2 microgram of total protein. The specificity of the detection is demonstrated by a comparison with the corresponding polypeptide profile obtained by silver nitrate staining of the gel.     

A novel method of electrophoresis for the separation of individual human serum glycoproteins

A method for the isolation of protein components by a combination of polyacrylamide gel electrophoresis and electro-extraction of slices of gel contained in cellophane dialysis tubing, using the H tube electrophoresis apparatus, is described. The method was applied to the purification of individual human serum glycoproteins. Although the glycoproteins have widely differing electrophoretic mobilities, they appeared to be antigenically interrelated judging from their complex "spurring" in Ouchterlony double gel diffusion tests.     

Alteration of glycoproteins during amphibian metamorphosis

In metamorphosing tadpole liver, the quantitative and qualitative changes in glycoproteins were observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and lectin-peroxidase method.     

Quantitation of glycoproteins on electroblots using the biotin-streptavidin complex

A method for the detection and quantitation of glycoproteins on nitrocellulose electroblots is described. Protein mixtures may be solubilized in sodium dodecyl sulfate prior to labeling, which is especially useful when dealing with membrane proteins. Mild periodate oxidation produces aldehydes on the oligosaccharide moieties which are then specifically condensed with biotin aminocaproyl hydrazide. After polyacrylamide gel electrophoresis and transfer of proteins onto nitrocellulose membranes, biotinylated glycoproteins are detected with enzyme-linked streptavidin and quantitated by densitometric scanning. As little as 1 ng of alpha 1-acid glycoprotein can be detected by this method. The use of mild oxidation conditions renders the method highly selective for the detection of sialic acid-containing glycoproteins.     

The staining of sciatic nerve glycoproteins on polyacrylamide gels with concanavalin A-peroxidase.

The plant lectin concanavalin A (Con A) possesses a remarkably specific capacity to bind primarily α-d-mannose or α-d-glucose sugar residues on macromolecules (cf. 1). The multivalent Con A will bind to carbohydrates on cell surfaces, and free binding sites on the attached Con A will bind to horseradish peroxidase which is a glycoprotein (2). Since peroxidase may be visualized by reaction with diaminobenzidine (3), it has been possible using this method to specifically “stain” carbohydrate residues on cell surface macromolecules (2, 4). The same principles for staining cell surfaces should apply to “staining” glycoproteins separated by polyacrylamide electrophoresis. In this paper, we examine the staining of glycoproteins in sciatic nerve by a Con A-peroxidase labeling technique. The method is more sensitive for mannose or glucose containing glycoproteins than the periodic acid-Schiff's (PAS) method commonly used.     

Detection of sialic acid containing compounds and the behaviour of gangliosides in polyacrylamide disc electrophoresis

A modified method is described for staining of sialic acid in glycoproteins and gangliosides in polyacrylamide gels using resorcinol reagent. The electrophoretic properties of ganglioside are discussed in view of the stability of their micelles in aqueous solutions.     

Detection of glycoproteins in the Acanthamoeba plasma membrane

In the present study we have shown that glycoproteins are present in the plasma membrane of Acanthamoeba castellanii by utilizing different radioactive labeling techniques. Plasma membrane proteins in the amoeba were iodinated by 125I-lactoperoxidase labeling and the solubilized radiolabeled glycoproteins were separated by lectin-Sepharose affinity chromatography followed by polyacrylamide gel electrophoresis. The periodate/NaB3H4 and galactose oxidase/NaB3H4 labeling techniques were used for labeling of surface carbohydrates in the amoeba. Several surface-labeled glycoproteins were observed in addition to a diffusely labeled region with Mr of 55,000-75,000 seen on electrophoresis, which could represent glycolipids. The presence of glycoproteins in the plasma membrane of Acanthamoeba castellanii was confirmed by metabolic labeling with [35S]methionine followed by lectin-Sepharose affinity chromatography and polyacrylamide gel electrophoresis.     

Staining of glycoproteins in polyacrylamide and agarose gels with fluorescent lectins.

We describe a simple and sensitive method for staining of the carbohydrate moiety of glycoproteins in polyacrylamide or agarose electrophoretic gels. Gels are incubated in a solution of fluorescein-labeled concanavalin A. Following destaining with a neutral buffer, glycoproteins exhibit fluorescence under long-range ultraviolet light. Thus, the glucose/mannose containing β- and γ-chains of human fibrinogen give fluorescent bands, whereas the carbohydrate-free α-chain does not react. Less than 100 ng of hexose bound to fibrinogen β- or γ-chains could be detected. The procedure is suitable for staining of other carbohydrate residues in glycoproteins, which can be recognised by specific agglutinins, as shown by binding of fluorescein-labeled lectins from Ricinus communis to galactose residues of fibrinogen.     

Microvilli membrane proteins from dog enterocytes]

Mcrovilli membranes have been isolated from dog jejunal and ileal enterocytes. Proteins were analysed by polyacrylamide gel electrophoresis after solubilization with sodium dodecylsulphate. The recovery of the membrane fraction with this purification method was found to be 22% and the specific activity of sucrase increases 19 folds in the membrane fraction. Microvilli membrane proteins after polyacrylamide gel electrophoresis were seprated in 21 bands, most of them with a molecular weight higher than 70 000. Seven bands with molecular weight from 150 000 to more than 340 000, were found to be glycoproteins.     

Human buccal epithelial cell receptors of Pseudomonas aeruginosa: identification of glycoproteins with pilus binding activity

Adherence of Pseudomonas aeruginosa to a patient's epithelial surface is thought to be an important first step in the infection process. Pseudomonas aeruginosa is capable of attaching to epithelial cells via its pili, yet little is known about the epithelial receptors of this adhesin. Using nitrocellulose replicas of polyacrylamide gels of solubilized human buccal epithelial cells (BECs), glycoproteins (Mz: 82,000, and four bands between 40,000 and 50,000) that bound purified pili from P. aeruginosa strain K (PAK) were identified by immunoblotting with a pilus-specific monoclonal antibody that does not affect pilus binding to BECs (PK3B). All pilus-binding glycoproteins were surface localized, as determined by surface radioiodination of intact BECs. Binding of pili to all of the glycoproteins was inhibited by Fab fragments of monoclonal antibody PK99H, which inhibits PAK pili binding to BECs by binding to or near the binding domain of the pilus, but not by Fab fragments of monoclonal antibody PK41C, which binds to PAK pilin but does not inhibit pili binding to BECs, demonstrating that pilus binding to these glycoproteins is likely via the same region of the pilus that binds to intact BECs. Periodate oxidation of the blot eliminated pili binding to all glycoproteins, indicating that a carbohydrate moiety is an important determinant for pilus-binding activity. However, not all of the glycoproteins exhibited the same degree of sensitivity to periodate oxidation. Furthermore, monosaccharide inhibition of pilus binding to BECs implicated L-fucose and N-acetylneuraminic acid as receptor moieties.     

Fluorescent staining of glycoproteins on polyvinylidene difluoride membrane with 8-aminonaphthalene-1,3,6-trisulfonate.

Here, we describe a simple and sensitive method that allows fluorescent detection of glycoproteins on polyvinylidene difluoride (PVDF) membrane. We used periodic acid oxidation of carbohydrate chains of glycoproteins and fluorescent labeling with 8-aminonaphthalene-1,3,6-trisulfonate (AN-TS) by reductive amination. We developed an additional method to enhance the ability of PVDF to absorb glycoproteins by using non-glycoprotein lectin, such as wheat germ agglutinin (WGA), as a link between the PVDF membrane and glycoproteins, resulting in considerably increased detection sensitivity to glycoproteins.     

The synthesis of fluorescent chlorotriazinylaminofluorescein-concanavalin A and its use as a glycoprotein stain on sodium dodecyl sulphate/polyacrylamide gels.

Concanavalin A, a specific glycoprotein probe, was optimally labelled to a maximum stoichiometry of 0.4 mol of chlorotriazinylaminofluorescein (CTAF)/mol of concanavalin A monomer under mild reaction conditions (pH 8.0, 6 h), and under these conditions the CTAF concanavalin A preparation retains its carbohydrate-binding ability and is able to penetrate SDS/7.5-15%-polyacrylamide gradient gels. CTAF-concanavalin A gives fluorescent bands for the glycoproteins transferrin, fetuin and deoxyribonuclease and shows no fluorescent response for the non-glycoproteins bovine serum albumin and soya-bean trypsin inhibitor. The detection limit of sensitivity for CTAF-concanavalin A, which is similar to that of fluorescein isothiocyanate-concanavalin A, is in the range 5-25 micrograms of glycoprotein. CTAF-concanavalin A is a suitable probe for the detection of glycoproteins in higher-percentage (greater than or equal to 10%) SDS/polyacrylamide gels, and will probably have other applications in, for example, fluorescent energy transfer and other structure-function studies.     

Sequential elution of proteins in small volumes by preparative polyacrylamide gel electrophoresis

A simple flexible method for separation of proteins by polyacrylamide gel electrophoresis and sequential elution into dialysis bags has been devised. The system was applied to isolation of three glycoproteins from the peritoneal fluid of mice bearing Ehrlich ascites tumor.     

FRACTIONATION OF SOLUBLE BRAIN GLYCOPROTEINS BY AFFINITY AND HYDROXYLAPATITE CHROMATOGRAPHY

—A separation of soluble brain proteins and Con A-binding glycoproteins by chromatography on calcium hydroxylapatite in the presence of SDS is described. Seventeen Coomassie Blue-stained bands were detected by polyacrylamide gel electrophoresis in SDS of Con A-binding glycoproteins of the soluble fraction of rat brain: 16 of these were found by in vivo uptake of [³H]fucose to be fucosylglycoproteins. Hydroxylapatite chromatography yielded several glycoprotein pools, each of which was shown by gel electrophoresis to contain between 4 and 8 individual glycoproteins. Such pools were enriched in [³⁶H]fucose relative to the brain soluble fraction by factors of between 6 and 21. Preliminary experiments demonstrate that this method is also applicable to the fractionation of membrane-bound glycoproteins.     

Two-dimensional polyacrylamide-gel electrophoresis of the proteins and glycoproteins of the human erythrocyte membrane.

A two-dimensional polyacrylamide gel electrophoresis technique has been developed, improving the analytical separation of some proteins and glycoproteins of the human erythrocyte membrane. Freshly prepared membranes are totally solubilized, subjected to dodecylsulfate--polyacrylamide gel electrophoresis in the first dimension, followed by electrophoresis in the second dimension, using a detergent-free polyacrylamide gradient gel. By this method the proteins of the human erythrocyte membrane could be resolved into a two-dimensional pattern, which has been shown to be highly reproducible with respect to various blood-groups and within one blood-group from specimen to specimen. The method enables especially the investigation of the hydrophobic and very likely integrated membrane proteins and glycoproteins. Thus, band III[Fairbanks, G., Steck, Th. & Wallach, D. F. H., Biochemistry, 10, 2606--2617 (1971)] could be shown to consist of five proteins, one of them being the major glycoprotein of the human erythrocyte membrand. The two spectrin bands differed considerably in their two-dimensional patterns. The value of the given method for the investigation of membrane defects, which may be linked with various diseases of human erythrocytes, could be demonstrated in the case of two patients suffering from congenital dyserythropoetic anaemia.     

Urinary glycoproteins in acute leukemias: a 41 000 dalton glycoprotein follows the kinetic of cytoreduction

Glycoproteins of leukemic cells and 24-hour urinary proteins were subjected to SDS polyacrylamide gel electrophoresis followed by affinity labelling I125 with Concanavalin A, indicating glycoproteins with mannose and/or glucose carbohydrate residues. Among the cellular glycoproteins a 41 000 dalton glycoprotein appeared under induction therapy in close correlation to the reduction of leukemic cells in ALL as well as in AML.