Anti-FTIP3/FTIP4 | FT Interacting Protein 3/4

Samples:
mctp3 mctp4 - Arabidopsis thaliana double knockout mutant
mctp3 - Arabidopsis thaliana single knockout mutant
mctp4 - Arabidopsis thaliana single knockout mutant
Wild type - Arabidopsis thaliana wilde type

10 µg od protein/well of total protein extracted freshly from 7 days old Arabidopsis thaliana seedlings. Exact buffer components were 150 mM Tris-HCl, pH 7.5; 150 mM NaCl; 10 % glycerol; 10 mM, EDTA, pH 8; 1mM NaF; 1 mM Na2MoO4; 10 mM DTT; 0.5 mM PMSF; 1% (v/v) P9599 protease inhibitor cocktail (Sigma); 1 % (v/v) Igepal) by incubating during 40 min at 4ºC with continuous mixing in an end-over-end rocker. Samples were centrifuged for 20 min at 4ºC and 9 000 g. Supernatants were filtered by gravity through Poly-PrepChromatography Columns and denatured at at 60ºC/20 min in Laemmli buffer (Tris-HCl pH 6.8 125 mM; 4% SDS; 20 % (v/v) glycerol; 2 % (v/v) beta mercaptoethanol; 0.01 % bromophenol blue) for SDS page loading. Samples were separated in the cold on 10 % SDS-PAGE and blotted for 1 h to nitrocellulose membrane (pore size of 0.2µm), using: wet transfer in the cold. Blot was blocked with 5 % nonfat milk in TBS-T at 4ºC/ON with agitation. Following the washes, the blot was incubated in the primary antibody at a dilution of 1: 500 for 1h/RT with agitation in TBS-T. The antibody solution was decanted, and the blot was rinsed twice, then washed four times for 10 min with TBS-T at RT with agitation. Blot was incubated in matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1: 10 000 in for 45 min/RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagent. Exposure time was 30 seconds.

Note: background signal can be decreased,  by lowering protein load/well and increasing primary antibody dilution.