A lectin method for investigating the glycosylation of nanogram amounts of purified glycoprotein.
To unravel the complexities of the glycosylation of a protein is a substantial task, which requires considerable effort and resources. However, in many situations this is unnecessary, because only a limited amount of information is required. A new lectin-binding assay is described which is rapid, cheap and versatile. A purified glycoprotein is absorbed on to the plastic surface of a microtitre plate. After removing unbound protein by washing, uncoated sites on the plate are blocked and digoxigenin or biotin-labelled lectin is added. The degree of lectin binding is measured using either an anti-DIG antibody or streptavidin conjugated enzyme, which is subsequently used to develop a colour reaction. Using this method it is possible to screen multiple specimens with high sensitivity and excellent precision. In addition, very small amounts of lectin are used, background absorbances are low, and the procedure does not require a high degree of technical skill. Because very small amounts of glycoprotein are needed, a glycoprotein can often be rapidly purified by batch affinity chromatography. The method has been successfully applied to several purified proteins using the lectins, Con A, LCA, LTA, MAA, and SNA, and the information obtained agrees with that produced by more sophisticated approaches, eg Dionex Carbohydrate Analyser. Using a panel of lectins, a carbohydrate structural profile is quickly built-up, and subtle differences in glycosylation identified. This method should be particularly useful for screening glycosylation in multiple clinical specimens; in specimens where very small amounts of material are available, such as membrane molecules; and in the screening of recombinant proteins produced commercially.
Goodarzi
M T
MT
Department of Clinical Biochemistry, The Medical School, Newcastle upon Tyne, UK.
Turner
G A
GA
D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006023:Glycoproteins / Q000032:analysis* / Q000302:isolation & purification; D006031:Glycosylation; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D006023:Glycoproteins