The use of ultrathin‐layer polyacrylamide gel isoelectric focusing in two‐dimensional analysis of plant and fungal proteins

Abstract

Although use of ultrathin polyacrylamide gel isoelectric focusing in the first dimension of two-dimensional analysis bestows a number of advantages, it has been little used by the plant science community. Nonstandardization along with problems unique to the format have probably delayed wider adoption. Relevant parameters were therefore tested in order to optimize resolution, reproducibility, economy and ease of use. Ultrathin-layer gels (200 microns in this study) used in the first dimension require a semirigid backing for support. Widely available matte-finished thin polyester film without chemical pretreatment was found to bind the gel adequately. The gel adheres to the film through all processing steps, yet, if desired, can be easily transferred to Whatman 3MM paper for special applications such as Western blotting. The ultrathin first-dimensional gels can be quickly dried on the polyester backing for convenient handling and long term storage. Strips cut from the dried gel for use in the second dimension are more easily manipulated than their tube format counterparts. The difficulty of disrupting and recovering microsamples of labeled leaf and root tissue prompted the invention of an efficient and simple communition device. An economical and efficient silver stain process is also described. This analytical technique was applied in an attempt to detect resistance gene products in different genetic backgrounds of maize. Although the ultrathin flatbed format provides as good as or better resolution than the tube gel system, the level of sensitivity was still inadequate to reveal the apparently rare resistance gene product.