BIN2 | Brassinosteroid insensitive 2

Samples: Lane 1 = Marker;  2 = 2.5 ng BIN2‐GST; 3 = 5 ng BIN2‐GST; 4 = 10 ng BIN2‐GST; 5 = 20 ng BIN2‐GST; 6 = 30 ng BIN2‐GST

From 2,5‐30 ng of total protein from recombinant BIN2‐GST purified protein eluted with the buffer 150 mM NaCl, 5mM DTT, 20mM GSH, 50mM TRIS/HCL pH 8.0 and denatured with 4X SDS +ß mercaptoethanol at 95°C for 5 min. The samples were separated on 10% SDS‐PAGE and blotted for 120 min to PVDF membrane using semi‐dry transfer. Blots were blocked with 5% milk powder in TBST for 60 min at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 5000 for 16 h overnight 4°C with agitation. The antibody solution was decanted, and the blot was rinsed briefly twice, then washed 1 time for 10 min in TBST buffer at RT with agitation. Blot was incubated in secondary antibody (anti‐rabbit IgG horse radish peroxidase conjugated, AS09 602, Agrisera) diluted to 1:10 000 in for 120 min at RT with agitation. The blot was washed as above and developed for 10 min with chemiluminescence detection reagent and exposed to X‐ray film. Exposure time was 60 seconds.
BIN2 ≈ 43 kDa GST ≈ 26 kDa BIN2‐GST ≈ 69 kDa

Courtesy of Dr. Pablo Altertos, Technical University of Munich, Germany