Magnetic bead purification as a rapid and efficient method for enhanced antibody specificity for plant sample immunoblotting and immunolocalization

Molecular analysis of plant tissues with antibodies has traditionally been hindered by the presence of high non-specific binding by plant cell walls a nd other components along with significant contaminants within sera that re tard identification of specific plant tissue targets. Methods which rely on immobile solid supports conjugated with high-affinity molecular entities, have been used to purify sera. Despite their wide use, traditional antibody purification methods can result in low yields or activity and can produce significant levels of secondary contaminants, resulting in high non-specifi c background and dilution of tissue-specific signals. Mobile support matrix es like magnetic beads conjugated with high-affinity antisera have recently become an efficient alternative method for isolating and identifying diver se molecular targets. In this study, rabbit anti-calreticulin (CRT) immunog lobulin G (IgG) was isolated from whole anti-CRT sera with magnetic beads a nd tested by Western blot and immunocytochemistry for CRT localization in P istia stratiotes plant tissues. IgG protein quantitation and purity was com pared between purified and non-purified pre-immune and anti-CRT sera using spectrophotometric, reducing sodium dodecyl sulfate-polyacrylamide gel elec trophoresis (SDS-PAGE). and fluorescence staining followed by quantitative densitometry analysis. Anti-CRT IgG binding specificity after purification was determined by Western blot of total soluble protein extract. Purified a nd non-purified pre-immune and anti-CRT samples were subsequently utilized for CRT immunogold localization in Pistia tissue sections and visualized wi th confocal microscopy. The results demonstrate that magnetic bead purified anti-CRT IgG from whole serum shows enhanced specificity and reduced backg round. The ease of use and speed of this IgG purification technique should find widespread use in the plant biology field. (C) 2000 Published by Elsev ier Science Ireland Ltd. All rights reserved.