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.
Holloway
P J
PJ
State Chemistry Laboratory, Department of Agriculture and Rural Affairs, East Melbourne, Victoria, Australia.
Arundel
P H
PH
D000665:Ampholyte Mixtures; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D001467:Hordeum / Q000032:analysis; D007525:Isoelectric Focusing; D017830:Octoxynol; D010455:Peptides / Q000032:analysis; D010940:Plant Proteins / Q000032:analysis*; D011092:Polyethylene Glycols; D012639:Seeds / Q000032:analysis
D000665:Ampholyte Mixtures; D010455:Peptides; D010940:Plant Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol