Western blot analysis
For immunoblot analysis, total protein was extracted from 4-week-old
leaves of plants (Jilani et al., 1996). Protein concentration of the
supernatants was measured, as described by Bradford (1976).
Electrophoresis was carried out on a 15% (w/v) SDS-PAGE gel using a
total of 25 µg of soluble plant protein per lane. Separated polypeptides
were blotted on nitrocellulose membranes (Jilani et al., 1996). Proteins
were probed with a primary antibody from Imgenex (India). The rabbit
anti-mouse IgG (1:25,000) was used as a secondary antibody, conjugated
to alkaline phosphatase. Blots were stained for alkaline phosphatase,
using 5-b romo-4-c hloro-3-i ndolylp hosphate (BCIP) and n itro b luet etrazolium (NBT) and quantified using an Alpha Imager 3400.