AKIN ß gamma
AS09 463 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana
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#1: 5μg of purified (Ni-NTA, Qiagen, manufacturer’s instruction) recombinant protein expressed in E. coli were loaded.
#2: Protoplast sample, AKINbg-HA was overexpressed as in Ramon et al (2013, the Plant Journal) and boiled in Laemmli solubilisation buffer for 5min. The proteins were separated on 8% SDS-PAGE and blotted 1h to PVDF using semi-dry transfer. Blots were blocked with 5% non-fat Milk in TBS for 1h at room temperature (RT) with agitation. Blots were incubated in the primary antibody at a dilution of 1:1000 for 12h at 4°C with agitation. The antibody solution was removed and the blot was then washed four times for 5 min in TBS-T at RT with agitation. Blots were incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:10 000 in 1% non-fat milk-TBS for 1h at RT with agitation. The blots were washed as above, 5 times, and developed for 5 min with ECL according to the manufacturer's instructions. Exposure time was 20 seconds for the recombinant proteins (#1) or 5 min for protoplasts sample (#2).
Courtesy of Dr Pierre Crozet and Dr Elena Baena-Gonzalez. Spain
It has not been confirmed working on endogenous cell extract.
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