Anti-CRT3 | Calreticulin-3

1 – 50 µg of Arabidopsis thaliana Col-0 wild type seedling extract 2 – 50 µg of Arabidopsis thaliana crt1 crt2 mutant seedling extract 3 – 50 µg of Nicotiana benthamiana leaf extract (mock infiltrated with empty vector control)
4 – 50 µg of Nicotiana benthamiana leaf extract transiently expressing A. thaliana CRT2-RFP fusion protein
5 – 50 µg of Nicotiana benthamiana leaf extract transiently expressing A. thaliana CRT3-HA fusion (HA-tag)
6 – 50 µg of Nicotiana benthamiana leaf extract transiently expressing A. thaliana CRT3-HA fusion (HA-tag, higher OD600 used for infiltration than in 5)

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 CRT3 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 a following chemiluminescent detection reagent: AgriseraBright. Exposure time was 10 seconds (Fusion FX, Vilber). For Nicotiana benthamiana samples: Leaves were extracted with RIPA buffer (Sigma Aldrich). The other experimental details were the same as for A. thaliana.

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

Alternative name: Protein PRIORITY IN SWEET LIFE 1