Mature Brassica seeds were ground into a fine powder in liquid nitrogen using mortar and pestle. Subsequently, 5 mL extraction buffer (50 mM Tris-HCl, pH 8, 500 mM sucrose, 200 mM NaCl, 0.1% Tween 20, 10 mM beta-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 5 mM ethylenediamine tetraacetic acid (EDTA)) was added to each gram of powdered seeds. The total soluble protein (TSP) was removed from cell debris by centrifugation of the seed extracts for 20 minutes at 4◦C (14000 rpm). Protein concentrations were determined by Bradford assay (42 (link)) using bovine serum albumin (BSA) as standard.
For western blot analysis, non-transgenic and transgenic seed protein extracts were loaded to a 15% SDS-acrylamide gel and by the end of electrophoresis protein bands were transferred to the polyvinylidene difluoride (PVDF) membrane (Millipore). The membrane was blocked with 5% (w/v) skimed milk in PBS-Tween (1/1000v/v) and after three washing steps incubated with anti-5xHis antibody (Qiagen, Germany) 1:10000 in PBS-T for two hours at room temperature. Following washing steps, membrane was incubated with diluted (1:8000) horseradish peroxidase -conjugated goat anti-mouse antibody (Sigma, USA) for one hour. Finally, tetramethylbenzidine (TMB) was used to develop the color.
For western blot analysis, non-transgenic and transgenic seed protein extracts were loaded to a 15% SDS-acrylamide gel and by the end of electrophoresis protein bands were transferred to the polyvinylidene difluoride (PVDF) membrane (Millipore). The membrane was blocked with 5% (w/v) skimed milk in PBS-Tween (1/1000v/v) and after three washing steps incubated with anti-5xHis antibody (Qiagen, Germany) 1:10000 in PBS-T for two hours at room temperature. Following washing steps, membrane was incubated with diluted (1:8000) horseradish peroxidase -conjugated goat anti-mouse antibody (Sigma, USA) for one hour. Finally, tetramethylbenzidine (TMB) was used to develop the color.