V cholerae

Example 5

α-Biotin Immunoblotting:

Aliquots collected during enrichment procedure (10 μL) were reduced and separated by standard SDS-PAGE (Bio-Rad, Criterion system), electroblotted onto nitrocellulose, blocked in 5% bovine serum albumin (Sigma) in Tris-buffered saline with Tween (10 mM Tris pH 8., 150 mM NaCl, 0.1% Tween-20), and analyzed by standard enhanced chemiluminescence immunoblotting methods (Pierce). Staining agent used: streptavidin-HRP (Pierce, 1:100,000).

Lectin Staining:

GalNAz-labeled or DMSO vehicle Jurkat media (100 μg) in buffer (25.0 μL, 50 mM NaOAc pH 5.5, 4 mM CaCl₂) was aliquoted in duplicate. One aliquot from each condition was treated with neuraminidase (4.0 μL, V. cholerae, Roche). Aliquots were mixed and incubated at 37° C. for 12 h. Aliquots (10 μL) were reduced and separated by standard SDS-PAGE (Bio-Rad, Criterion system), electroblotted onto nitrocellulose, blocked in Tris-buffered saline with Tween (10 mM Tris pH 8, 150 mM NaCl, 0.5% Tween-20), and analyzed by standard fluorescent imaging (Typhoon 9410, GE Healthcare). Staining agent used: peanut agglutinin-FITC (Vector Laboratories, 1:100).

Example 5

α-Biotin Immunoblotting:

Aliquots collected during enrichment procedure (10 μL) were reduced and separated by standard SDS-PAGE (Bio-Rad, Criterion system), electroblotted onto nitrocellulose, blocked in 5% bovine serum albumin (Sigma) in Tris-buffered saline with Tween (10 mM Tris pH 8, 150 mM NaCl, 0.1% Tween-20), and analyzed by standard enhanced chemiluminescence immunoblotting methods (Pierce). Staining agent used: streptavidin-HRP (Pierce, 1:100,000).

Lectin Staining:

GalNAz-labeled or DMSO vehicle Jurkat media (100 μg) in buffer (25.0 μL, 50 mM NaOAc pH 5.5, 4 mM CaCl₂) was aliquoted in duplicate. One aliquot from each condition was treated with neuraminidase (4.0 μL, V. cholerae, Roche). Aliquots were mixed and incubated at 37° C. for 12 h. Aliquots (10 μL) were reduced and separated by standard SDS-PAGE (Bio-Rad, Criterion system), electroblotted onto nitrocellulose, blocked in Tris-buffered saline with Tween (10 mM Tris pH 8, 150 mM NaCl, 0.5% Tween-20), and analyzed by standard fluorescent imaging (Typhoon 9410, GE Healthcare). Staining agent used: peanut agglutinin-FITC (Vector Laboratories, 1:100).