Exponential phase bacterial cultures in CO2IM were pelleted and concentrated 100 times in saline or saline + 0.05 M methyl-mannopyranoside (Sigma). For yeast agglutination, 25 µl of the saline or saline + 0.05 M methyl-mannopyranoside resuspension were transferred on a microscopy glass slide and mixed with either an equal volume of fresh baker’s yeast suspension in saline (15 mg/ml) or saline only (negative control) for 30 s. For blood agglutination, 25 µl of the saline resuspension was transferred on a microscopy slide and mixed with an equal volume of a 2% suspension of isolated human red blood cells in saline or with saline only (negative control). Results for both yeast and blood agglutination were observed macro- and microscopically.
Methyl mannopyranoside
Manufactured by Merck Group
Methyl mannopyranoside is a carbohydrate compound used as a biochemical research tool. It is a derivative of the monosaccharide mannose, with a methyl group attached. The compound is often utilized in studies involving carbohydrate-protein interactions and as a component in various analytical techniques.
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3 protocols using methyl mannopyranoside
1
Bacterial Agglutination Assay
2
Purification of HIV-1 Envelope Proteins
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HIV-1 Envelope proteins were made and purified as described previously (Pujanauski et al., 2013 (link)). Monomeric gp120 (ADA) and trimeric gp140 (YU2) were produced by transient transfection of COS7 cells (ATCC) with 5 µg of gp120 or gp140 plasmid (provided by T.M. Ross, University of Georgia, Athens, GA) and Lipofectamine (Invitrogen). gp120 and gp140 were purified as before (Pujanauski et al., 2013 (link)), using a column made with agarose-bound lectin (Vector Laboratories). HIV-1 Envelope protein, bound to the column, was washed using PBS, and eluted with 1M methyl mannopyranoside (Sigma-Aldrich). The eluted protein was buffer exchanged and concentrated into sterile PBS using 15 ml capacity centrifugal 30-kD cutoff filters (Ambicon). Protein purity was checked by Western blot with murine anti-gp120 antibody (ImmunoDx) and SDS-PAGE.
3
Production and Purification of HIV-1 Trimeric gp140
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Trimeric gp140 (YU2) was produced as previously described (34 (link)). In brief gp140 was generated by transient transfection of COS7 cells (ATCC) using 5 μg of gp140 plasmid (gift from T.M. Ross, University of Georgia, Athens, GA) and Escort IV Transfection Reagent (Sigma-Aldrich). Purification of gp140 was achieved using a column made with agarose-bound Galanthus nivalis lectin (Vector Laboratories). Gp140 was bound to the column, washed with PBS, and eluted using 1M methyl mannopyranoside (Sigma-Aldrich). The purified protein in eluant was buffer exchanged into sterile PBS and concentrated using Vivaspin 20 centrifugal filters with a 30-kD cutoff (Sartorius). Protein purity was checked by SDS-PAGE. Purified gp140 was stored at −20°C and aliquoted to prevent multiple freeze/thaw cycles.
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