GLYCAN DETECTION BY LECTIN BLOTTING

University Of Georgia, Athens GA

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Lectin blotting was carried out using DIG glycan differentiation kit (Roche). Briefly, the sample and controls were blotted onto the nitrocellulose membrane (1 [unreadable]g of the sample, positive and negative control. The membranes were immersed in a blocking solution (supplied by the kit) followed by incubation with Digoxigenin-labeled lectins (Table 1) 1[unreadable]g/ml in TBS. The binding activity was visualized using 750 mU alkaline-phosphatase-conjugated sheep anti-Digoxigenin as secondary antibody and nitro blue tetrazolium/5-bromo-4-chloro-3-indoyl phosphate as color developing reagent. Carboxypeptidase Y (a, GNA positive), Transferin (b, SNA positive), Fetuin (c, MAA positive) and Asialofetuin (d, PNA and DSA positive) were used as positive controls. Bovine serum albumin (BSA) was used as a negative control. Ponceau S staining was used for detection of protein on the membrane.