Anti ugpase polyclonal antibody

Total protein extracts were prepared from 3-day-old etiolated seedlings maintained in darkness or irradiated with 20 μmol m⁻² s⁻¹ of blue light for 15 min. Under a dim red safe light, plant tissue was ground in a mortar and pestle in extraction buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton-X 100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride (PMSF) and a protease inhibitor mixture (Complete EDTA-free; Roche) and clarified by centrifugation at 10,000 g, 4°C for 10 min. The resulting supernatant was used as the total protein extract. Protein concentrations were determined by the Bradford colorimetric method (Bio-Rad). All samples were mixed with SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.004% bromophenol blue), boiled for 4 min and subjected to 7.5% SDS-PAGE. Proteins were transferred onto polyvinylidene fluoride (PVDF) membrane (GE Healthcare) by electroblotting and detected with anti-phot1 polyclonal antibody (Cho et al., 2007 (link)), anti-NPH3 polyclonal antibody (Tsuchida-Mayama et al., 2008 (link)), and anti-UGPase polyclonal antibody (Agrisera). Blots were developed with horseradish peroxidase (HRP)-linked secondary antibodies (Promega) and Pierce ECL Plus Western Blotting Substrate (Thermo Fisher Scientific).

Total proteins were extracted from 3‐day‐old etiolated seedlings by directly grinding 100 seedlings in 100 μl of 2× SDS sample buffer. Dissection of seedlings into apical and basal segments was performed under a dissecting microscope with micro scissors (Fine Science Tools, Heidelberg, Germany) with red safe light illumination. Proteins were transferred onto polyvinylidene fluoride membrane (GE Healthcare, Little Chalfont, UK) by electroblotting and detected with anti‐phot1 polyclonal antibody (Cho et al., 2007), anti‐NPH3 polyclonal antibody (Tsuchida‐Mayama et al., 2008) and anti‐UGPase polyclonal antibody (Agrisera, Vännäs, Sweden). Blots were developed with horseradish peroxidase‐linked secondary antibodies (Promega, Southampton, UK) and Pierce ECL Plus Western Blotting Substrate (Thermo Fisher Scientific, Renfrew, UK).

Total proteins were extracted from 3-d-old etiolated seedlings by directly grinding 50 seedlings in 100 µL of 2× SDS sample buffer under red safe light illumination. Light-grown tissue was frozen and ground to a fine powder in liquid nitrogen before 150 mg of tissue was mixed with 100 µL of 2× SDS sample buffer. Proteins were transferred onto nitrocellulose or polyvinylidene fluoride membrane and detected with anti-phot1 or anti-phot2 polyclonal antibodies (8 (link)), anti-NPH3 polyclonal antibody (58 ) and anti-UGPase polyclonal antibody (Agrisera). Blots were developed with horseradish peroxidase-linked secondary antibodies (Promega) and Pierce ECL Plus Western Blotting Substrate (Thermo Fisher Scientific).