Anti-DOG1 alpha splice variant

Samples
wt: Arabidopsis thaliana
wild type dog1-3: Arabidopsis thaliana knockout of DOG1 gene
dog1-5: Arabidopsis thaliana increased expression of DOG1 protein

25 µg/well of total protein extracted freshly from dry seeds of Arabidopsis thaliana. Were denatured at 90°C/5 mintues. Samples were separated in the cold on 10 % SDS-PAGE and blotted for 1 h to PVDF membrane (pore size of 0.45 µm), using: wet transfer in the cold. Blot was blocked with 5 % milk in TBS-T at 4°C/ON with agitation. Blot was incubated in the primary antibody at a dilution of 1: 2 000 in 5 % milk in TBS-T for ON/4°C with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1: 20 000 in 5 % milk in TBS-T for h/RT with agitation. The blot was washed as above and developed with a fchemiluminescent detection reagent: AGFA G153 Developer (REF HT536, Agfa Corporation) and Carestream Dental X-ray Fixer (REF 5060694, Carestream Health). Exposure time was 7 minutes.