Elimination of artifacts on native western blots arising from endogenous lectin activity.
While studying the behavior of profilin from Phaseolus vulgaris seeds under native conditions, a high molecular weight species suggesting a complex of profilin and associated proteins was observed by Western immunoblotting. This putative complex was also observed when enzyme-linked secondary antibodies alone were used, and this apparently resulted from antibody association, through its glycosyl moieties, with the endogenous carbohydrate-binding activity from the seed extracts. This endogenous activity corresponded to that of purified phytohemagglutinin (PHA). In addition, the P. vulgaris lectin activity was very stable and was observed when the extracts were pretreated with varying concentrations of sodium dodecyl sulfate, Triton X-100, urea and beta-mercaptoethanol, or when membrane blots were boiled in water before incubation with antibody. The activity was abolished only if the membrane was boiled in 1% sodium dodecyl sulfate. This finding could also be useful to implement assays for carbohydrate-binding activity from cell or tissue extracts using different visualizable reagents bearing particular glycosyl moieties.
Villanueva
Marco A
MA
Instituto de Biotecnología, U.N.A.M., PO Box 510-3, Cuernavaca, Morelos 62250, Mexico. marco@ibt.unam.mx
D000818:Animals; D000906:Antibodies; D015153:Blotting, Western / Q000379:methods*; D003285:Contractile Proteins; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D007120:Immunochemistry; D037102:Lectins / Q000276:immunology / Q000302:isolation & purification*; D008840:Microfilament Proteins / Q000276:immunology / Q000302:isolation & purification; D027805:Phaseolus / Q000737:chemistry; D010835:Phytohemagglutinins / Q000276:immunology / Q000302:isolation & purification; D037121:Plant Lectins; D051304:Profilins; D012639:Seeds / Q000737:chemistry
D000906:Antibodies; D003285:Contractile Proteins; D037102:Lectins; D008840:Microfilament Proteins; D010835:Phytohemagglutinins; D037121:Plant Lectins; D051304:Profilins