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13. SUPPLEMENTARY MATERIALS
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Preparation of plant material
Approximately 1-2 leaves and a pinch of purified silica sand were put into plexi mortars.
For all enzymes but peroxidases, two drops of 0.35% 2-mercaptoethanol were added as the
extraction buffer. In a case of peroxidases deionized H 2
O was used. The plant material was
ground into fine, homogenous and viscous powder with a glass pestle. Wicks of 5 mm by 10
mm were soaked in the leaf extract and protected from drying by covering with plant material.
The material was kept at 4 o C until use (approximately 15-20 minutes). Then soaked wicks
were transferred into the narrow slit in the gel.
Electrophoresis conditions
Lithium borate/Tris citrate buffer system and 11% starch gel were used. Electrophoresis
was run at 4 o C at constant power up to the moment the front had reached 8 cm from the
origin (Table 13.3.2). Once the electrophoresis had been completed, the gel was chilled and
then cut into 5-6 slices depending on number of staining enzymes. Each slice was placed in
a different stain box and was stained for a single enzyme.
Staining protocols
The distance of migration of specific proteins through the starch gel was visualized by
histochemical staining (Table 13.3.3). The own modifications of protocols described by Zielinski
(1987) were adopted. Each stain consisted of a substrate, on which a specific enzyme
reacts, and dyes or substances that fluoresce under UV light. Some stains also contained
cofactors, coupling enzymes and other molecules. All enzymes were stained in 100 ml of
staining solution because this technique gave clearer and stronger bands than an agar overlay
method. In addition the bands were durable and the gels could be stored without loss of
quality in a refrigerator for a long period. All stains but peroxidases were incubated at 37 o C
until the bands were clearly visible. Peroxidases were stained at room temperature. After
staining, the gel was fixed in a methanol, deionized water and glacial acetic acid (60%) by
a ratio of 5:5:1 for at least 1 h, covered with plastic food wrap and refrigerated.