LFY | Leafy
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1 : 1000 (WB)
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20 µg of total protein: Markers (1), 35S:: LFY lines tsp1 (2), 35S:: LFY lines E1 (3), 35S:: LFY lines tsp2 (4), 35S:: LFY lines E2 (5), Empty well (6), 35S:: LFY lines tsp3 (7), 35S:: LFY lines E3 (8), LFY recombinant protein (9) extracted freshly from leaves with IP buffer (Co-IP kit from Invitrogen) and denatured with SDS reducing dye at 90°C for 5 min were separated on 12 % SDS-PAGE and blotted for 10 min to PVDF using dry transfer system (iBlot, Invitrogen). Blot was blocked with blocking buffer for 10 min at RT with agitation. Blot was incubated with primary antibody (anti-LFY, Agrisera) at a dilution of 1: 1 000, washed, incubated with Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, AS09 602 Agrisera) diluted to 1:25 000 and washed. The incubations were performed for 2h 30 min using the ibind system (Invitrogen). The blot was developed for 1 min with ECL reagents (BioRad). Exposure time was 100 seconds. The red arrow indicates the position of LFY from in planta samples and the blue the position of the truncated version of recombinant LFY.
Courtesy of Dr. Claudius Marondedze, CEA, France
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