Anti-EDS1 | Enhanced disease susceptibility 1

Samples:
1 -40 ug of Arabidopsis thaliana whole leaf extract, non treated
2 -40 ug of Arabidopsis thaliana whole leaf extract, treated with PstAvrB 3h
3- 40 ug of Arabidopsis thaliana whole leaf extract, old-6 weeks
4-40 ug of Solanum lycopersicum (negative control) 8 discs extract, control leaf
5-40 ug of Solanum lycopersicum (negative control) 8 discs extract, wounded leaf
6-40 ug of Arabidopsis thaliana whole leaf extract, mutant eds1
7-40 ug of Arabidopsis thaliana whole leaf extract, mutant eds1
Mark: MW markers: 5uL Thermo scientific pageruler plus prestained ladder

40 ug/well of total protein was extracted freshly from Arabidopsis thaliana and Solanum lycopersicum (see above) with a buffer: Tris-HCl pH 7,5 50mM; 10% glycerol; 1mM EDTA; 200mM NaCl; 1mM DTT; 0,1% Triton X-100; Sigma protease cocktail; sigma phosphatase inhibitor cocktail; 140 mM pepstatine A and 0,1mM PMSF in MiliQ water (samples 1-5) and Tris-HCl 50mM pH 7.8; 0.2% triton x-100; Sigma protease inhibitor cocktail; 1% PMSF and 1.5% pepstatin A in MiliQ water; (samples 6-7) and denatured with SDS buffer (Tris-HCl pH 6,8, 8% SDS, 40% glycerol) and 1ul DTT 0,5 M at 95°C for 5 min. Proteins were separated on 12 % SDS-PAGE and blotted 1h to PVDF (pore size of 0,45 µm), using semi-dry. Blot was blocked with 3 % non-fat milk for 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 with agitation in 3% milk-TBS-T 0,1 %T ON/4°C. The antibody solution was decanted and the blot then washed three times for 10 min 3% milk-TBS-0,1%T at RT with agitation. Blot was incubated in Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated AS09 602) diluted to 1:10 000 in 3% milk-TBS-0,1%T for 1h/RT with agitation. The blot was washed three times wit TBS and developed for 4 min (300uL + 300 uL / membrane) with either Agrisera ECLSuperBright. Exposure time was 1800 seconds in total. The image correspond to 1660-second exposure.

Courtesy of Dr. María C. Romero-Puertas, Department of Biochemistry, Cell and Molecular Biology of Plants, CSIC, Spain