Con A-Sepharose

Analyses Crude supernatant Con-A Sepharose eluant Recovery... 

Affi-Gel Protein A Protein A Superose Protein G Superose Con A Sepharose Protein A Immunoglobulin G BIORAD Pharmacia Pharmacia Pharmacia... 

Human-platelet membranes have been extracted with lithium 3,5-diiodosalicylate, and the soluble glycoproteins separated on O-phosphonocellulose.849 The purified, membrane glycoprotein, molecular weight 100,000, was immunochemically identical to a sample purified from platelet-membrane extract by means of con A-Sepharose affinity chromatography. No further characterization was reported,... 

A mixture of lentil lectin-reactive glycoproteins from pig lymphocyte-plasma membrane was isolated by lentil lectin-Sepharose chromatography of sodium deoxycholate-solubilized membranes.445 Eighty-seven percent of the protein applied (17% of hexose) passed through unretarded, and 13% of the applied protein (83% of hexose) was bound, and eluted with methyl a-D-glucopyranoside solution. Recovery was 95% of the material applied, in contrast to the recovery in similar experiments conducted on con A-Sepharose columns (80% recovery).850 The eluate from the lentil column, which contained at least ten glycoproteins, blocked lymphocyte transformation induced by lentil or kidney-bean lectins.445... 

A water-soluble, 50%-methanol-soluble, acidic glycoprotein was isolated from rat brain by affinity chromatography on con A-Sepharose.855 The glycoprotein, pure by poly(acrylamide) gel-electrophoresis at pH 8.8, had an apparent molecular weight of 14,500 1,400 by dodecyl sodium sulfate gel-electrophoresis. No analysis for carbohydrate was reported.855... 

The principal application of lectins in bioanalytical systems involves the reversible immobilization of glucose oxidase, invertase, and peroxidase on Con A-Sepharose. Such lectin-based affinity media have also been utilized for immobilization of glycoenzymes. Woodward (18) shows that cellobiase is not desorbed by its substrate cellobiose and product glucose from the support matrix. 

Murthy and Herez described a procedure for the purification of a-antitrypsin based on affinity chromatography on Con A-Sepharose [131]. The glycoprotein a,-fetoprotein has been purified on Con A-agarose [132]. Affinity chromatography of a,-fetoprotein on Con A-agarose has been used to demonstrate two molecular variants of the protein and was used in the resolution of these two forms [133]. 

Allan, Auger and Crumpton used Con A-Sepharose to isolate glycoprotein cell surface receptors for concanavalin A from pig lymphocyte membranes solubilized with sodium deoxycholate [134]. 

The application of insolubilized lectins in polysaccharide fractionation was demonstrated by Kennedy and Rosevear with Con A-Sepharose... 

Mixtures of polysaccharides were separated on Con A-Sepharose by differential elution, weakly interacting polysaccharides were eluted with phosphate buffer, and more tightly bound polysaccharides were removed with boracic buffer. 

The second approach [54] was based on the inhibition of acetylcholine esterase. One unit of acetylcholine esterase was reversibly immobilized via lectin binding to Con A-Sepharose and could be rinsed off with a pulse of 0.2 M gly-cine-HCl, pH 2.2. Reversible immobilization of enzymes and whole cells in the enzyme thermistor column, utilising specific lectin-glucoprotein interactions, had been introduced earlier and was especially useful for inhibition studies, where the enzyme had to be replaced very often. Enzyme activity was determined with 10 mM butyrylcholine as a substrate. A 5 -10 min pulse of pesticide solution was introduced into the flow buffer, followed by a second substrate pulse. The decrease in activity was proportional to the amount of pesticide, with a detection limit below 1 ppm. 

Con A-Sepharose is commercially supplied as a suspension in Con A-bufTer and 0.02% merthiolate. The bed volume of the Con A-Sepharose column should be 1 ml per 3 mg of POase. The column should be rinsed prior to use with about 5 volumes of PBS-C or Con A buffer. The passage of free IgG is monitored at 278 nm. After all IgG passed, POase complexes are desorbed with a-methyl-D-mannopyranoside, at 10-100 mM in PBS-C or Con A buffer. This sugar competes with POase at the binding sites, and needs not to be removed from the conjugate prior to El A. The difference in detectabilities in EIA using conjugates with or without free IgG is considerably more than expected from relative concentrations of free IgG and conjugate (Tijssen et al., 1982). 

Partially purified serologically actiye fractions, ACI-B, MHI-B and RNI-B, were separated by affinity chromatography with a con A sepharose column from the supernatant of disrupted mycelium fluid of cyl indrospora, JM. hiemal is and J,. nigricans, respectively. These active fractions contained mannose as their... 

An anionic serum inhibitor of Clq was separated from a partially purified Clq preparation using Con A-Sepharose. The inhibitor exhibited Clq hemolytic activity in a dose-dependent fashion (Conradie et al., 1975) and formed a relatively insoluble inhibitor-Clq complex. The chemical nature of this Clq inhibitor is not known. Certain anionic polymers are known to bind with Clq and inhibit Cl hemolytic activity liquoid, dextran sulfate, polyvinylsulfate, and heparin (Raepple et al., 1976). Thus molecules that bind to Clq may or may not activate Cl, indicating that a specific binding activity is necessary for activation. 

Cathepsins H from different organs and species were found to have molecular weights of 26,000-28,000. The higher p7 value of this enzyme (6.5-7.5) than of cathepsin B and L forms the basis of its separation by DEAE-cellulose chromatography. Cathepsin H is a glycoprotein and binds to Con A-Sepharose 12). 

All the lysosomal thiol proteinases described in Section II have been isolated only recently and few structural studies have been carried out. The amino acid composition of only cathepsin B has been reported and is quite similar to those of cathepsin B from human liver (75) and rat liver (19) however Ouchterlony double difPiision analysis with antiserum against rat liver cathepsin B showed no immunological cross reactivity (40). All the lysosomal enzymes examined were shown to contain carbohydrate. The 111 of Asn is a glycosylation site in rat liver cathepsin B (128) but no detailed analysis of this carbohydrate has been reported. Since human liver cathepsin H can be purified on Con A-Sepharose (12), it may also contain a carbohydrate moiety. Rat liver cathepsin H and L also bind to Con A-Sepharose. 

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