AHB1 | Non-symbiotic hemoglobin 1 (A. thaliana)
AS18 4230 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana
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90 µg/well of total protein extracted freshly from Arabidopsis thaliana seedlings with extraction buffer (100mM Tris-HCl, 150mM NaCl, 0.25% NP-40) containing 1mM PMSF and 1X cOmplete® EDTA-free proteases inhibitors, Sigma) and denatured with buffer (Tris-HCl 0,25 M, pH 6,8; SDS 8% (w/v); glicerol 40% (v/v); β-Mercaptoethanol 20% (v/v) and Bromophenol Blue) at 90°C for 10 min were separated on 12% SDS-PAGE and blotted (20min; 2A; 25V) to PVDF (pore size of 0.2 µm), using semi-dry transfer. Blot was blocked with 5% milk for 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1000 ON/4°C with agitation. The antibody solution was decanted and the blot was washed 2 times for 5 min in TBS-T at RT with agitation. Blot was incubated in Agrisera matching secondary antibody (Goat anti-rabbit IgG HRP peroxidase conjugated, AS09 602) diluted to 1:25 000 in for 1h/RT with agitation. The blot was washed 4 times for 5 min in TBS-T at RT with agitation and developed with Agrisera ECL SuperBright, AS16 ECL-S. Exposure time was 5 seconds.
The lines used in the experiments were generated by:
-Hebesltrup et al., 2008 corresponds to 35S: GFP-PGB1
-Perazzolli et al., 2004 corresponds to 35S:PGB1
Courtesy of Dr. Inmaculada Sánchez-Vicente and Isabel Manrique-Gil, USAL-CIALE Salamanca, Spain
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