Double electrophoretic separation and lectin analyses of the component chains of secretory immunoglobulin A from human saliva.
A new method is presented for the separation of secretory immunoglobulin A (SIgA) from salivary samples. Salivary proteins (from parotid or stimulated whole mouth saliva) were precipitated with methanol to concentrate SIgA from salivary samples whilst removing other salivary proteins. SIgA purified from breast milk and salivary proteins was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions. Following completion of electrophoresis the top strip of gel was removed and the proteins present reduced with dithiothreitol. The gel strip was then applied to the top of a second 10% SDS gel, and the proteins were electrophoresed and then stained by Coomassie Brilliant Blue R-250. Three major protein bands were stained in all samples corresponding in molecular mass to secretory component, alpha-heavy chain and light chains of SIgA. Separated proteins were also electroblotted onto nitrocellulose and stained by fluorescein isothiocyanate (FITC). Lectin analysis was then used to detect the O-glycans present on IgA1. Lectins from Helix aspersa and Arachis hypogaea were used to determine the amount of terminal N-acetyl galactosamine and nonsialylated O-glycans, respectively. Maclura pomifera lectin was used to determine the total amount of IgA1 present on the blots. The results indicate that SlgA in stimulated whole mouth saliva, stimulated parotid saliva and purified from breast milk contain similar O-glycans.
Carpenter
G H
GH
Secretory and Soft Tissue Research Unit, Guy's, King's and St Thomas' School of Dentistry, The Rayne Institute, London, UK. guy.carpenter@kcl.ac.uk
Proctor
G B
GB
D000328:Adult; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D006801:Humans; D007071:Immunoglobulin A, Secretory / Q000032:analysis* / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000032:analysis; D007147:Immunoglobulin Light Chains / Q000032:analysis; D037102:Lectins / Q000378:metabolism; D008297:Male; D000432:Methanol; D019887:Peanut Agglutinin / Q000378:metabolism; D011134:Polysaccharides / Q000032:analysis*; D011485:Protein Binding; D012463:Saliva / Q000276:immunology*; D013194:Staining and Labeling / Q000379:methods
C038095:Helix lectin; D007071:Immunoglobulin A, Secretory; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D037102:Lectins; D019887:Peanut Agglutinin; D011134:Polysaccharides; D000432:Methanol