GalNAz-labeled or DMSO-vehicle Jurkat conditioned medium (100 μg) in buffer (25.0 μL, 50 mM NaOAc, pH 5.5, 4 mM CaCl2) was divided into two aliquots. One aliquot from each condition was treated with neuraminidase (4.0 μL, Vibrio cholerae, Roche). Aliquots were mixed and incubated at 37 °C for 12 h. Aliquots (10.0 μL) were reduced and separated by standard SDS-PAGE (Bio-Rad, Criterion system), electro-blotted onto nitrocellulose, blocked in PBS with 0.5% Tween-20, and analyzed by standard fluorescent imaging (Typhoon 9410, GE Healthcare). The staining agent was peanut agglutinin–FITC (Vector Laboratories, 1:100).
Peanut agglutinin fitc
Manufactured by Vector Laboratories
Peanut agglutinin-FITC is a plant lectin that binds to terminal galactose or N-acetylgalactosamine residues on cell surfaces. It is conjugated to the fluorescent dye fluorescein isothiocyanate (FITC) to enable visualization and detection of cells expressing the appropriate carbohydrate ligands.
Automatically generated - may contain errors
3 protocols using peanut agglutinin fitc
1
Analysis of Glycosylation Profiles
2
Biotinylated Protein Analysis via Immunoblotting
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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).
3
Biotin and Lectin Immunoblotting Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
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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).
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