A simple, rapid and quantitative method for preparing Arabidopsis protein extracts for immunoblot analysis.
Although Arabidopsis has numerous well documented advantages for genetic and molecular analyses, its small size can be a limitation for biochemical and immunochemical assays requiring protein extraction. We have developed a rapid method to extract total protein from small amounts of Arabidopsis tissue that can be used for quantitative immunoblot analysis. The procedure involves direct extraction of tissue into SDS-containing buffer under conditions permitting immediate protein quantification in the extract, using commercially available kits without prior fractionation. This approach provides maximal extraction and quantitative recovery of total cellular protein, together with accurate evaluation of target protein levels as a proportion of the total. We have examined the utility and sensitivity of the procedure using monoclonal antibodies to phytochromes A and C (phyA and phyC), which are high- and low-abundance members, respectively, of the phytochrome family in 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 single leaf from 3-week-old adult plants. The data indicate that the procedure may have broad utility for the detection and quantitative analysis of many proteins, including those of low abundance, in a variety of applications in Arabidopsis. In one such application, we used transgenic Arabidopsis phyC-overexpressor seedlings to demonstrate that the procedure can be used to detect transgene-encoded protein early at the segregating T2 generation, thereby offering the capacity for accelerated screening and selection of lines engineered to overexpress target proteins.
Martínez-García
J F
JF
Department of Plant and Microbial Biology, University of California, Berkeley 94720, USA.
Monte
E
E
Quail
P H
PH
D017360:Arabidopsis / Q000737:chemistry*; D029681:Arabidopsis Proteins; D015151:Immunoblotting; D010834:Phytochrome / Q000737:chemistry*; D051942:Phytochrome A
D029681:Arabidopsis Proteins; C497973:PHYA protein, Arabidopsis; D051942:Phytochrome A; D010834:Phytochrome