Anti-AHB1 | Non-symbiotic hemoglobin 1 (A. thaliana)

Samples:
1 - 90 ug of Arabidopsis thaliana whole seedling extract, Col-0 (wild type) submerged to induce PGB1
2 - 90 ug of Arabidopsis thaliana whole seedling extract, Col-3 (wild type) submerged to induce PGB1
3 - 90 ug of Arabidopsis thaliana whole seedling extract, Col-0; 35S: GFP-PGB1 in air (control)
4 - 90 ug of Arabidopsis thaliana whole seedling extract, C24; 35S:PGB1 in air (control)
5 - 90 ug of Arabidopsis thaliana whole seedling extract, C24; 35S:PGB1 submerged to induce PGB1
6 - 90 ug of Arabidopsis thaliana whole seedling extract, Col-0 (wild type in air (control)
7 - 90 ug of Arabidopsis thaliana whole seedling extract, C24 (wild type) in air (control)
8 - 90 ug of Arabidopsis thaliana whole seedling extract, knock down mutant of PGB1 (C24 background) submerged to induce PGB1
9 - 90 ug of Arabidopsis thaliana whole seedling extract, knock out mutant of PGB1 (Col-3 background) submerged to induce PGB1 (AHB1)

Mark: MW markers: Dual Color (BioRad)

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