High-resolution two-dimensional electrophoresis of plant proteins.

A technique for the analysis of plant proteins from seed, leaf, root, and coleoptile tissues by high resolution two-dimensional electrophoresis is described. This technique is based primarily on the procedure of P. O'Farrell (1975, J. Biol. Chem. 250, 4007-4021); however, a number of improvements and simplifications have been introduced. We have found that resolution of polypeptides from a range of plant tissues is improved if the concentrations of nonionic detergent and ampholytes used in the isoelectric focusing (IEF) step are increased to 4 and 5% (w/v), respectively. Further increase in the concentrations of these two components results in gels of decreased resolution and low mechanical strength. We have also found that substitution of n-octyl beta-D-glucopyranoside or 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate for Triton X-100 or Nonidet-P40 in the IEF dimension significantly increases the resolution of polypeptides in these gels. This technique also allows minor polypeptide differences between closely related cultivars of plants to be identified.

Optimal conditions for the use of cDNA probes to measure the concentration of barley yellow dwarf virus in barley (Hordeum vulgare).

Experiments which optimise the conditions for the measurement of the relative concentration of BYDV in barley (Hordeum vulgare) tissues using cDNA probes are described herein. These studies have shown that both the pH of the buffer and the ratio of buffer to tissue used to homogenise plant material greatly affects the amount of cDNA probe which hybridises to leaf extracts immobilised on nitrocellulose. These studies also showed that the measurement of this virus was greatly facilitated by using a dot-blot apparatus which allows samples contact with a piece of nitrocellulose 10 mm in diameter rather than a 3 mm (approx) diameter piece of nitrocellulose as is the case with most commercial dot-blot apparatuses. Further experiments using this technique showed that there was a large difference in the rate of replication of the PAV, BYDV serotype between BYDV-resistant and BYDV-susceptible cultivars of barley. These data suggest that a BYDV-resistant cultivar can easily be distinguished from a BYDV-susceptible one if the BYDV content of leaves is measured between 7 and 14 days after inoculation.