RGA | DELLA protein RGA
AS11 1630 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana
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64 | 64 kDa
40 µg of total protein from 5-d-old dark-grown Arabidopsis thaliana seedlings extracted with 50 mM Tris-HCl pH 7.5,, 10% glycerol, 150 mM NaCl, 0.1% NP-40, 1 mM PMSF, and 1x protease inhibitor cocktail (Roche) were separated on 4-20 % SDS-PAGE and blotted 1h to PVDF. m: mock, P: paclobutyrazol, P+G: PAC+GAs. Blots were blocked with 2% blocking reagent (GE Healthcare) in TBS-T for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 for 1h at RT 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. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from Agrisera, AS09 602 ) diluted to 1:10 000 in for 1h at RT with agitation. The blot was washed as above and developed for 5 min with chemiluminescence detection reagent, according to the manufacturers instructions. Exposure time was 20 seconds in a LAS-3000 Imager (Fuji).
Courtesy of Dr. David Alabadi, IBMCP (CSIC-UPV), Valencia, Spain
Courtesy of Kamila Jaronczyk, IBB, Warsaw, Poland
RGA protein is prone to degradation therefore caution has to be taken during protein extraction.
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