Figure 2 .4 Scatchard plot (Scatchard, 1 949) for antiserum raised against zeatin-riboside and used in radioimmunoassay. The slope of the curve gives an estimate of the affinity constant (K). Non-linearity is common with polyclonal antiserum due to a heterogeneous population of antibodies. The maximum and minimum slopes (- - -) were estimated for the range of the standard curve.
Cross reactivity of antiserum to naturally occurring and synthetic cytokinins was tested in RlA (Table 2.2) and these were found to be similar to those found in other l aboratories I see B adenoch-lones et al. ( 1 987) for review}. Of the cytokinins tested for cross reactivity, only the corresponding free base for each cytokinin (zeatin and isopentenyl adenine) showed appreciable cross reactivity (Table 2.2). Logit transformed standard curves for the free base cytokinins had parallel slopes to ribosyl cytokinins (Figure 2.8).
2- 1 4 2: General Materials and Methods. Cytokinin Analysis Table 2.2 Molar cross reactivity of anti-zeatin-riboside sera or anti-isopentenyl-adenosine sera. Cross reactivity were determined at the concentration of compound which bound 50% of the radioactivity bound in the presence of nil cytokinin and represents
moles cytoki nin equi \tIlents detected
moles compound added Compound trans-Zeatin-riboside trans-Zeatin cis-Zeatin Dihydrozeatin Dihydrozeatin-riboside Isopenteny I-adenosine Isopentenyl-adenine 6-Furfurylarninopurine (Kinetin) 6-Benzylaminopurine Adenosine Adenine
N-(2-chloro-4-pyridyl)-N' -phenyl urea (CPPU)
Zeati n-ri boside Isopenteny ladenosine
antiserum antiserum 1 00 1 . 1 40 0.3 0.6 0.8 1 .0 0.2 1 .3 0.2 3. 1 1 00 0.9 37.6 0.5 0.9 0.5 3 . 2 0 0 0 0 0 0
2: General Materials and Methods . Cytokinin Analysis
2.2.4 Radioimmunoassay (RIA) of ZR and IPA
2- 1 S
Polypropylene micro-centrifuge tube ( I.Sml, Sarstedt OS I S88) were tested for their binding of 3H-cytokinin dialcohols and 3H-ABA. Absorption of 3H-tracers from a buffer solution, was found to be not significantly (P<O.OO1 ) above background counts and these tubes were used as RIA reaction tubes.
Phosphate buffered saline (PBS) was prepared by dissolving KH2P04 0.38g, K2HP04 1 .3g, NaCI 8.78g in I I millipure water and adjusted to pH 7.4. For preparation of 1 00% saturated NH4S04, a super saturated solution was prepared by dissolving N�S04 to saturation in boiling water and allowing excess NH4S04 to precipitate as the solution cooled to room temperature. 90% saturated N�S04 was then prepared by diluting 9 parts saturated NH4S04 with 1 part H20 and adjusting pH to 7.4.
Tritiated dialcohol in methanol (20 000 disintegrations per minute (DPM), 1 00/-LI) was added to cytokinin sample or standard in an assay tube and taken to dryness in the speedvac. PBS (400J.ll) containing antiserum (diluted to bind 50% of 3H-dialcohol) and SJ.llfml new-born calf serum was added and the lid closed. The addition of the entire reaction mixture at once, rather than the standard method of separate addition of buffer and antiserum (Ernst, 1 986) enabled fewer pipette operations and therefore reduced between tube variation, but did not affect the assay.
Tubes were allowed to pre-incubate at room temperature for 20 minutes to allow the 3H_ dialcohol and cytokinin to re-dissolve, after which tubes were vortexed briefly to mix and incubated for a further 40 minutes. Binding of 3H-tracer by antiserum reached equilibrium after 40 minutes and was insensitive to total incubation time between 40 and I SO minutes (Figure 2.S). A 60 minute total incubation period was used for routine assay for convenience and to allow for slight differences in timing between tubes. Retention of radioactivity by the tube was approximately 300 OPM or 1 .5 % of radioactiv ity added following the 20 minute pre-incubation period and was not influenced by total incubation time up to I SO minutes.
2- 1 6 2: General Materials and Methods. Cytokinin Analysis To precipitate the antibody and bound tracer, 500 III of 90% saturated NH4S04 was added to bring tubes to a final 50% NH4S04 saturation. Tubes were vortex mixed and incubated at room temperature for a further 20 minutes. Tubes were then placed into a 24 place fixed angle micro-centrifuge (Heraeus Biofuge) with hinges facing out and centrifuged for 3 minutes at 1 3000 RPM (- 1 0000 x g). The fixed angle causes the precipitate to be coated along the wall facing outwards (below hinge). Although temperature was not controlled in the micro-centrifuge, the rotor was well insulated from the spindle and consequently there was no appreciable rise in temperature. A syringe needle attached to a vacuum was slid down the wall of the tube opposite to the hinge to aspirate the entire supernatant. The pellet was re-dis solved in 200lli methanol, mixed with 1 ml In-Flow 3 scintillation fluid (INfUS Systems Inc., Fla.) and measured for radioactive emission on the scintillation counter. Radioactive emission was automatically corrected for counting efficiency using the external standard method and was recorded as DPM. Duplicate measurements of DPM were taken for each tube.
Non-specific binding (NSB) was measured as the amount of tracer retained in the absence of antiserum. In the absence of antiserum, NSB of the tracer was usually less than 4% of the total tracer added. This was able to be reduced by approximately 0.5% by washing the tube with 50% saturated N�S04, but this was not considered necessary for routine assay. Up to I SO minutes after addition of buffers, NSB was insensitive to total incubation time (Figure 2.S). Perlstein ( 1 987) points out the NSB does not always remain constant over the entire concentration range. However NSB was not found to be affected by the addition of cytokinin at concentrations present in the standard curve, although it was significantly lower (contrast, p=O.OOOS) when cytokinin was present in vast excess ( 1 0 000 ng). This was due to significantly lower (contrast, P=0.0007) permanent binding of the tracer to the tube in the presence of an excess of cytokinin. The inability of standard curve range cytokinins to inhibit tracer binding to the tube suggests that either the tracer itself, or that breakdown products of the tracer had a higher affinity for binding sites on the tube than cytokinin. As the tracer which had degraded (see next paragraph) showed increased levels of NSB, it is possible that tube-bound radioactivity consisted of breakdown products rather than dialcohoI .
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