A simple, rapid and quantitative method for preparing Arabidopsis protein extracts for immunoblot analysis

Although Arabidopsis has numerous well documented advantages for genetic an d molecular analyses, its small size can be a limitation for biochemical an d immunochemical assays requiring protein extraction. We have developed a r apid method to extract total protein from small amounts of Arabidopsis tiss ue that can be used for quantitative immunoblot analysis. The procedure inv olves direct extraction of tissue into SDS-containing buffer under conditio ns permitting immediate protein quantification in the extract, using commer cially available kits without prior fractionation. This approach provides m aximal extraction and quantitative recovery of total cellular protein, toge ther with accurate evaluation of target protein levels as a proportion of t he total. We have examined the utility and sensitivity of the procedure usi ng monoclonal antibodies to phytochromes A and C (phyA and phyC), which are high- and low-abundance members, respectively, of the phytochrome family i n Arabidopsis. Both phytochromes could be rapidly and readily quantified in the tissues examined, with phyC being detectable in extracts representing as few as five dark-grown seedlings, two light-grown seedlings, or half a s ingle leaf from 3-week-old adult plants. The data indicate that the procedu re may have broad utility for the detection and quantitative analysis of ma ny proteins, including those of low abundance, in a variety of applications in Arabidopsis. In one such application, we used transgenic Arabidopsis ph yC-overexpressor seedlings to demonstrate that the procedure can be used to detect transgene-encoded protein early at the segregating T-2 generation, thereby offering the capacity for accelerated screening and selection of li nes engineered to overexpress target proteins.