Phly | DNA photolyase (At4g25290)
AS15 2864 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana
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78 | 90 kDa
2,5 µg of total protein from Arabidopsis thaliana wilde type darkness (1), wilde type light (2) and insertion mutants: SALK_056328C darkness (3), SALK_056328C light (4), extracted with 0.1 M Tris-HCl pH 8.5, 4% SDS, 2% (v/v) 2-mercaptoethanol, 2 mM phenylmethylsulfonyl fluoride and denatured with Laemmli buffer at 95oC for 10 min were separated on 12% SDS-PAGE and blotted 2h to PVDF using semi-dry transfer. Blots were blocked with 5% milk PBS-T (Tween 0.5%) for 30 min. at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1:1000 overnight at 4°C with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed 3 times for 10 min in 5% milk PBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from Agrisera) diluted to 1:25 000 in 5 %milk PBS-T for 1h at RT with agitation. The blot was rinsed briefly twice, then washed 3 times for 10 min in PBS-T at RT with agitation. Blot was developed with chemiluminescent detection reagent. Exposure time was 5 minutes.
Courtesy of Dr. Justyna Łabuz, Department of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Poland
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