Anti-CRT1/2 | Calreticulin-1/2

Samples:
1 – 50 µg of Arabidopsis thaliana Col-0 wild type seedling extract
2 – 50 µg of Arabidopsis thaliana crt1 crt2 mutant seedling extract

50 µg/well of total protein extracted freshly from A. thaliana seedlings. Exact buffer components were: 1 x PBS supplemented with 1 % (v/v) Triton X-100 and denatured with Laemmli buffer at 95 °C for 5 min. Samples were separated at room temperature by 10 % SDS-PAGE and blotted for 1 h to a nitrocellulose membrane (pore size of 0.45 µm), using wet transfer with a cooling block. The blot was blocked with 5 % (w/v) milk in TBS-T for 1 h/RT with agitation. The blot was incubated in the primary CRT1/2 antibody at a dilution of 1:1000 in TBS-T overnight at 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. The blot was incubated in matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:30000 in TBS-T for 2h/RT with agitation. The blot was washed as above and developed with the following chemiluminescent detection reagent: AgriseraBright. Exposure time was 20 seconds (Fusion FX, Vilber).

Courtesy Dr. Richard Strasser, Department of Applied Genetics and Cell Biology, BOKU, Austria