Ecl western blot detection and analysis system

The presence of the TaAGC1-overexpressing transgene in the transformed wheat plants was monitored by PCR using the specific primers, TaAGC1-1584F (located in TaAGC1 coding sequence) and Tnos-R (in Tnos of the transformation vector). PCR was performed in a 20-μl volume containing 200ng genomic DNA, 1×PCR buffer (TaKaRa), 0.4 μM each primer, 200 μM each dNTP and 1U Taq polymerase (TaKaRa). The amplified product (386bp) specific to the introduced TaAGC1-Tnos chimera was resolved on a 1.5% agarose gel and visualized by ethidium bromide staining.
The myc-TaAGC1 fusion protein in the overexpressing transgenic wheat lines was visualized by western blotting analysis. Total proteins were extracted from 0.3g of ground leaf powder. Total soluble proteins (~12 μg) for each line were separated on 12% SDS-PAGE and transferred to polyvinyl difluoride membranes (Amersham Biosciences). The western blots were incubated with 100-fold diluted anti-c-myc antibody followed by secondary antibody conjugated to horseradish peroxidase (GE Healthcare). The myc-TaAGC1 proteins were visualized using the ECL Western Blot Detection and Analysis System (GE Healthcare).

For protein expression analysis, 0.2 g of fresh leaf tissue was excised from transgenic T₀ plants and non-transgenic plants and homogenized in 500 μL of extraction buffer containing 50 mM of 1 M Tris–HCl (pH 6.8), 0.2% (w/v) polyvinylpyrrolidone (PVP-40) and 1% (v/v) β-mercaptoethanol. Samples were stirred for 30 min at 4°C and then clarified by centrifugation at 10,000 g. The protein content in the crude extract was determined by the Bradford method [19 (link)], using bovine serum albumin as standard. For each plant, approximately 50 μg of crude protein extract was subjected to 12% (w/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. The presence of the SnOLP protein was detected using polyclonal antibody specific for tobacco osmotin (kindly supplied by Dr. Bernard Fritig and Dr. Pierrette Geoffroy, Institut de Biologie Moléculaire des Plantes du C.N.R.S, France). The protein bands were visualized using the ECL Western Blot Detection and Analysis System (GE Healthcare). To disrupt less-specific interaction more stringent conditions were used by including detergent (0.1% Tween-20) in the wash solution.