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
2
.
1
. Total soluble protein extraction P. stratiotes water lettuce shoot tips were har-
vested, weighed, and frozen in liquid nitrogen. Pistia tissues where then ground to a fine pow-
der with mortar and pestle, after which two vol- umes of extraction buffer 25 mM Tricine, 10 mM
b-mercaptoethanol, 1 wv polyvinylpolypyrro- lidone PVPP, 1 mM EGTA, pH 7.5 were added
per gram of tissue. One micromolar leupeptin, 1 mM pepstatin A, and 10 mg100 ml phenylmethyl-
sulfonyl fluoride PMSF proteinase inhibitors were added to the extraction buffer just prior to
use. Resultant extract was centrifuged at 17 000 × g for 10 min at 4°C, the supernatant collected and
Pistia total soluble protein concentration deter- mined using the Pierce Coomassie method Pierce,
Rockford, IL.
2
.
2
. Magnetic bead IgG purification Magnetic bead antibody purification was per-
formed as previously published [16] with some modifications. Briefly, two 500-ml polystyrene-
coated paramagnetic iron bead slurry aliquots pre- conjugated with sheep anti-rabbit IgG antibody
M-280 Dynabeads; Dynal, Lake Success, NY were washed with 2 × 500 ml cold phosphate-
buffered saline PBS-T; 10 mM sodium phos- phate, 150 mM NaCl, 0.1 Tween-20, pH 7.4.
Suspended beads were collected after each wash with a magnetic particle collector Dynal MPC-E;
Dynal, Lake Success, NY. Bead aliquots were subsequently incubated with 7.5 ml crude rabbit
pre-immune or anti-CRT serum diluted in 250 ml cold PBS-T for 1 h at room temperature under
constant bidirectional mixing Dynal Sample Mixer 947.01; Dynal, Lake Success, NY. Bead
pellets were then washed with 3 × 500 ml cold PBS-T and collected as described above. Pre-im-
mune or anti-CRT rabbit IgG was eluted from magnetic beads with 500 ml 0.5 M acetic acid, pH
4.5 for 15 min at room temperature under con- stant bidirectional mixing. Beads were again col-
lected with a magnetic particle collector, then supernatant removed from the immobilized bead
pellet and immediately neutralized with 500 ml 1 M Tris – HCl, pH 8.5. Eluant protein concentration
was determined by dividing A
280
readings collected from a Shimadzu UV-240 UV-Visible Recording
Spectrophotometer Shimadzu, Kyoto, Japan by a protein extinction coefficient of 1.4. Neutralized
eluant was subsequently centrifuge-concentrated via a 30-kDa-cutoff Centricon spin concentrator
Millipore, Bedford, MA. Final pre-immune and anti-CRT concentrates were stored at 4°C.
2
.
3
. IgG purity and densitometry analysis Pre-immune and anti-CRT IgG content in crude
whole sera was determined using 1:1 and 1:2 whole serum diluted in 10 mM Tris – HCl, 150 mM
NaCl, pH 7.2 TBS, separated on a 12 acry- lamide SDS-PAGE gel under reducing conditions,
and visualized using Coomassie staining. Pre-im- mune and anti-CRT IgG were compared to a
non-specific IgG standard. Percent IgG was visu- ally determined and multiplied by the dilution
factor to estimate average percent IgG.
The Laemmli method [17] was used with the following modifications. Two 0.75-mm, 10 SDS-
PAGE gels were run at 150 V 62 mA for 90 min under
reducing conditions,
then fluorescence
stained with Sypro Red FMC, Rockland, ME as follows. SDS-PAGE gels were briefly rinsed with
double distilled water ddH
2
O, then incubated with 1:5000 Sypro Red in 7.5 vv acetic acid in a
polypropylene container for 1 h at room tempera- ture under constant mixing. Gels were rinsed with
1 × 20 ml additional 7.5 acetic acid and stored in ddH
2
O prior to signal detection. Sypro Red-stained SDS-PAGE gels were excited
using a Molecular Dynamics Storm 860 laser scan- ner version 4.1; Molecular Dynamics, Sunnydale,
CA set to 530 nm connected to a Power Macin- tosh G3 300 MHz running Mac OS 8.1. Fluores-
cent signals
were visualized
with Molecular
Dynamics ImageQuant version 1.2; Molecular Dynamics, Sunnydale, CA software and exported
as tagged image format TIF files. TIFs were then imported into and quantitatively analyzed using
BioRad Quantity One densitometry software ver- sion 4.0.3; BioRad, Hercules, CA. Absolute pro-
tein amounts for magnetic bead purified pre-im- mune and anti-CRT gels were determined by
comparison to a six-point BSA standard calibra- tion curve using extrapolation and linear regres-
sion.
2
.
4
. Western blot analysis Total soluble proteins were separated on a 10
SDS-PAGE gel, stained with Sypro-Red and imaged as described above, then transferred to
Immun-Blot polyvinylidene difluoride PVDF membrane BioRad; Hercules, CA at 200 mA
constant current overnight. Following transfer, the membrane was rinsed briefly with TBS, then
non-specific protein binding sites blocked in 1 Western Blocking Solution WBS; Boehringer-
Mannheim; Indianapolis IN in TBS for 1 h at room temperature. Membrane was then incubated
with a 1:200 dilution of either magnetic bead purified anti-CRT IgG or anti-CRT whole serum
in 0.5 WBS in TBS for 2 h at room temperature. Incubated membranes were subsequently washed
with 2 × 100 ml TBS containing 1 vv Tween-20 TBS-T and 2 × 100 ml 0.5 WBS in TBS for 10
min under constant rotational mixing. Secondary antibody incubation of membranes followed with
1:5000 goat anti-rabbit IgG horseradish peroxi- dase HRP conjugate Sigma, St. Louis, MO in
0.5 WBS in TBS for 1 h at room temperature. Finally, the membrane was rinsed 4 × 10 min with
TBS-T, drained and briefly blotted, then incubated with chemifluorescent substrate ECL Plus Amer-
sham-Pharmacia; Piscataway, NJ under foil cover for 5 min at room temperature. Protein bands in
total soluble sample were imaged using a Molecu- lar Dynamics Storm 860 Laser Scanner set at 488
nm. Digital images were visualized with Molecular Dynamics ImageQuant, exported as TIF files and
quantitatively analyzed against a six-point BSA curve with BioRad Quantity One densitometry
software as described above.
2
.
5
. CRT immunolocalization Pistia shoot tips were dissected into 2 mm
2
sections and fixed in 4.0 paraformaldehyde in 50 mM [Piperazine, N, N-bis-2-ethane-sulfonic acid,
1.5 sodium salt] Pipes, pH 7.2 overnight at 4°C. Fixed tissue was dehydrated through a 30, 50, 70,
80, 95 and 100 increasing ethanol series, infiltrated with LR White acrylic resin EM Sci-
ences; Ft. Washington, PA and cured overnight at 60°C. One micrometer Pistia shoot tip sections
were mounted on silane-coated slides Digene Di- agnostics; Beltsville, MD, then non-specific bind-
ing sites blocked for 1 h in 10 mM Tris – HCl, 150 mM NaCl, 0.3 vv Tween-20, 1 wv bovine
serum albumin BSA, pH 7.2 TBS-TBSA. Sec- tions were incubated in 1:100 whole or magnetic
bead purified pre-immune or anti-CRT sera di- luted in TBS-TBSA for 4 h at room temperature.
Shoot tip sections were subsequently washed 4 × 15 min in TBS-TBSA, then incubated with 1:100
protein A-gold diluted in TBS-TBSA for 1 h at room temperature. Sections were sequentially
washed 2 × 15 min in TBS-TBSA, TBS-T, and ddH
2
O, then silver-enhanced and visualized on a BioRad MRC1000 laser scanning confocal micro-
scope BioRad; Hercules, CA. Reflected and transmitted tissue images were obtained then
merged to generate the final Pistia shoot tip sec- tion images.
3. Results
