Introduction Directory UMM :Data Elmu:jurnal:P:PlantScience:PlantScience_Elsevier:Vol153.Issue1.2000:

Plant Science 153 2000 7 – 14 Magnetic bead purification as a rapid and efficient method for enhanced antibody specificity for plant sample immunoblotting and immunolocalization Ian J. Quitadamo a,1 , Todd A. Kostman 1 b , Margaret E. Schelling a , Vincent R. Franceschi b, a School of Molecular Biosciences, Washington State Uni6ersity, Pullman, WA 99164 - 4234 , USA b School of Biological Sciences, Washington State Uni6ersity, Pullman, WA 99164 - 4236 , USA Received 1 October 1999; received in revised form 7 October 1999; accepted 7 October 1999 Abstract Molecular analysis of plant tissues with antibodies has traditionally been hindered by the presence of high non-specific binding by plant cell walls and other components along with significant contaminants within sera that retard 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-specific background and dilution of tissue-specific signals. Mobile support matrixes like magnetic beads conjugated with high-affinity antisera have recently become an efficient alternative method for isolating and identifying diverse molecular targets. In this study, rabbit anti-calreticulin CRT immunoglobulin G IgG was isolated from whole anti-CRT sera with magnetic beads and tested by Western blot and immunocytochemistry for CRT localization in Pistia stratiotes plant tissues. IgG protein quantitation and purity was compared between purified and non-purified pre-immune and anti-CRT sera using spectrophotometric, reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis 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 and non-purified pre-immune and anti-CRT samples were subsequently utilized for CRT immunogold localization in Pistia tissue sections and visualized with confocal microscopy. The results demonstrate that magnetic bead purified anti-CRT IgG from whole serum shows enhanced specificity and reduced background. The ease of use and speed of this IgG purification technique should find widespread use in the plant biology field. © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords : Antibody; Calreticulin; Immunocytochemistry; Magnetic bead; Microscopy; Purification www.elsevier.comlocateplantsci

1. Introduction

Calreticulin, a low-affinity, high-capacity cal- cium binding protein found in the endoplasmic reticulum ER of most eukaryotic cells [1 – 3], has been isolated from several plant species including barley, Euglena, Gingko and pea [4 – 7]. The exact roles of calreticulin CRT in plant systems is still in question and is the focus of much investiga- tion [8]. CRT is hypothesized to be an important component of the calcium buffering system found in the ER, but has also been reported to serve as an ER chaperone and signaling molecule, and to mediate protein – protein interactions [1]. We are particularly interested in the role of high capacity calcium binding proteins in calcium ox- alate formation in plants [9], which is a calcium buffering process unique to plants [10,11]. Im- munological techniques are critical to our analysis of the role of such proteins in the specialized cells that produce calcium oxalate. Corresponding author. Tel.: + 1-509-335-3025; fax: + 1-509-335- 3517. E-mail address : vfrancesmail.wsu.edu V.R. Franceschi 1 Co-contributors. 0168-945200 - see front matter © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 9 9 0 0 2 4 3 - 5 However, protein localization in plant tissue presents unique problems. Plant cells contain a multitude of components including pigments phe- nolics, polysaccharides and glycoproteins that non-specifically bind antibody serum protein com- ponents like immunoglobulin G IgG and serum albumin [12]. In particular, cell walls are a persis- tent source of antibody non-specific binding due the presence of complex polysaccharides [13] and lignins. The reactivity of plant cell wall and other components can make target protein localization by immuncytochemical techniques difficult. To al- leviate antibody non-specific binding, plant scien- tists employ a variety of measures. Common approaches include increasing blocking agent and or detergent concentrations as well as buffer strin- gency by increasing salt levels. However, these approaches may also decrease antibody specific binding to target proteins. Thus, a delicate balance must be struck between eliminating antibody non- specific binding and promoting antibody specific target recognition. An alternative to these approaches is antibody purification, which reduces serum secondary con- taminants. Traditional methods like affinity chro- matography are often employed for antibody purification; however these methods may produce low yields of active antibody [14] that often still contains significant amounts of secondary contam- inants [15,16]. An alternative method for enhanc- ing antibody purification and specificity in plant biology use involves magnetic bead purified anti- body [16]. Previous studies using magnetic bead purified antibodies have shown enhanced antigenic specificity and immunologic activity in mam- malian tissues [16]. As compared to other antibody purification methods like traditional affinity chro- matography, magnetic bead purification methods can produce antibodies of superior purity and activity [16]. Magnetic bead antibody purification is also a very rapid technique, but to our knowl- edge it has not been applied to plant biology work. For our research purposes, we have generated polyclonal antibody in rabbits to plant CRT. The purpose of this study was to analyze the effective- ness of magnetic bead imunoaffinity purification for enhancing CRT immunolocalization in Pistia stratiotes plant tissues. Sheep anti-rabbit IgG-con- jugated paramagnetic beads were utilized for a quantitative IgG purification scheme from whole pre-immune and anti-CRT sera. Following mag- netic bead purification of preimmune and anti- CRT IgG, antibody purity and immunologic activity were assessed using sodium dodecyl sul- fate-polyacrylamide gel electrophoresis SDS- PAGE and Sypro Red fluorescence staining in addition to Western blot analysis. Non-purified and purified pre-immune and anti-CRT IgG were subsequently used for CRT localization in Pistia plant tissue sections. We show that magnetic bead IgG purification is a quick and effective method for antibody preparation to be used on plant samples.

2. Materials and methods