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
3
.
1
. Serum and purified IgG protein determination Whole anti-CRT serum protein concentration as
determined by protein assays was 42 mgml. This value was used for total pre-immune serum
protein concentration as well. Magnetic bead purified pre-immune and anti-CRT IgG protein
A
280
readings were 0.017 and 0.014, with calcu- lated protein concentrations of 0.243 and 0.20
mgml, respectively. Absolute protein amounts and percent IgG yield via magnetic bead purifica-
tion are depicted in Table 1.
3
.
2
. BSA calibration cur6e The BSA standard calibration curve was deter-
mined by loading 0.01, 0.02, 0.04, 0.1, 0.2 and 1.0 mg known BSA quantities on a reducing 10
acrylamide SDS-PAGE gel and subsequently staining with Sypro Red. Known BSA quantities
produced 9.47, 20.3, 22.18, 36.45, 54.33, and 120.5 fluorescence signal densities, respectively. Plotted
values produced a BSA linear regression curve correlation coefficient of 0.9928.
3
.
3
. IgG analysis Analysis of whole pre-immune and anti-CRT
Table 1 Magnetic bead purification efficiency
a
Aliquot uses Serum IgG mg
Eluant IgG mg Purification efficiency
IgG antigen 1
15
b
12.8 85
Pre-immune 2
15
b
9.3 62
15 7
11.4 76
10 15
b
6.4 43
1 CRT
15
b
13.6 91
2 15
b
12.9 86
15 10.7
7 71
15
b
9.3 62
10
a
Magnetic bead uses refers to the number of times a single bead aliquot was used for purification. Pre-immune and anti-CRT bead aliquots were used specifically for same antibody isolation as previous purifications. Percent yield values were rounded to the
nearest whole percent.
b
Represents bead aliquots loaded six times over manufacturers stated bead capacity.
Fig. 1. SDS-PAGE of magnetic bead purified pre-immune A and anti-CRT B sera. Arrows indicate positions of IgG
heavy chain HC and light chain LC proteins. Samples include molecular weight marker MW, whole pre-immune
A or anti-CRT B serum diluted 1:30 Serum, supernatant collected following whole serum incubation with magnetic
beads Super, washes following serum incubation with mag- netic beads Washes 1 – 3, and centrifugal-concentrated pre-
immune or anti-CRT magnetic bead eluant product MB.
Fig. 2. A. Western blot of Pistia total soluble protein from −
80°C stored stock, incubated with either whole anti-CRT serum WS or magnetic bead purified anti-CRT IgG MB.
Whole serum gives multiple bands while bead purified IgG detects CRT and a breakdown product of CRT BDP. B.
Western blot of Pistia freshly prepared total soluble protein extract incubated with magnetic bead purified anti-CRT.
Breakdown product is very minor in non-frozen samples. BDP, Break down product.
albumin, IgG heavy and light polypeptide chain proteins, respectively. Whole anti-CRT serum di-
luted 1:30 contained 3.04, 1.83 and 0.82 mg, whereas supernatant collected following magnetic
bead incubation with undiluted whole serum con- tained 2.49, 1.48 and 1.09 mg albumin, IgG heavy
chain, and IgG light chain. Magnetic bead purified and concentrated sample contained 0.95 mg IgG
heavy chain and 0.91 mg IgG light chain, showing a 1:1 heavy:light chain ratio, which is as expected.
Similar values were obtained during identical anal- ysis of whole pre-immune serum, supernatant,
washes and magnetic bead purified pre-immune IgG samples data not shown. Magnetic bead
purified IgG had negligible contaminant serum protein as shown on gels using the very sensitive
Sypro Red detection Fig. 1A, B.
3
.
4
. Western blot analysis Western blot analysis of Pistia total soluble
protein extracts separated via reducing SDS- PAGE gels and electroblotted to PVDF mem-
branes Fig. 2A produced the following values when quantitatively analyzed against the BSA
standard curve. Blots incubated with whole sera were compared to blots incubated with magnetic
bead purified antibody eluant. Two bands were recognized in both blot preparations; a 66- and
45-kDa band. With whole serum, multiple non- specific bands were identified on the blot, which
did not show up with the purified protein. Using magnetic bead purified anti-CRT IgG, a band
with 1.38 mg protein was identified at 66 kDa in Pistia soluble extract. A band of identical size was
also found using whole anti-CRT serum, which detected 1.78 mg in Pistia soluble extract. An
additional band of 1.93 mg was observed in soluble extract at 45 kDa using magnetic bead purified
anti-CRT. Whole anti-CRT serum also produced a 2.34-mg 45 kDa band in Pistia soluble extract.
To determine if the 45-kDa band, seen in Fig. 2A, using magnetic bead purified ant-CRT was a CRT
breakdown product or cross reaction with another protein, a new Pistia soluble protein extract was
prepared and immediately run on a 10 SDS- PAGE gel, electroblotted to PVDF membrane,
and incubated with magnetic bead purified anti- CRT as described above. The new CRT blot also
showed the presence of two bands; however, the 66-kDa band was by far the most dense, with only
a faint secondary band visible Fig. 2B.
serum, supernatant, washes and magnetic bead purified samples via reducing SDS-PAGE, fluores-
cent stained gels Fig. 1A, B and quantitative comparison to the BSA calibration curve pro-
duced the following quantitative values for serum
3
.
5
. CRT immunolocalization Immunolocalization patterns were compared for
non-purified and purified pre-immune and anti- CRT IgG sera using confocal microscope imaging
of silver-enhanced immunogold labeling Fig. 3. Fig. 3A, B depict immunolocalization patterns
using whole pre-immune and anti-CRT sera re- spectively. Background is very high with both the
pre-immune and anti-CRT whole sera. In particu- lar, the vacuoles and crystals of crystal idioblasts
and the walls of many cells show labeling that is non-specific. In contrast as seen in Fig. 3C, the
purified pre-immune IgG gave completely clean sections with no wall or vacuolar labeling. With
purified anti-CRT IgG, labeling was restricted to the cytoplasm of all cells, and was particularly
abundant in the cytoplasm of crystal idioblasts Fig. 3D, while vacuolar and wall labeling were
essentially eliminated. The amount of specific label was also much higher with the purified anti-CRT
IgG as compared to the whole anti-CRT serum. Thus, magnetic bead purified CRT IgG gave
greatly increased signal to noise ratio for labeling on resin embedded sections, while background was
eliminated when purified pre-immune IgG was used.
4. Discussion
