AGO6 | Argonaute 6
AS10 672 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana
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116.4 | 99 kDa
360 µg/well of Arabidopsis thaliana protein extracted from leaf using TCA-protein precipitation (for method check protocol tab) and saturated in 8M urea were separated on 15% SDS-PAGE and blotted for 1hour to 0.2 µm nitrocellulose at 100V using wet transfer system. Blots were blocked with 0.5% cold fish gelatin for 1hr at room temp with agitation. Blot was incubated in the primary antibody (anti-H3) at a dilution of 1:2500 for an hour at RT with agitation. The blots were washed with 3X 15min TBS-TT at RT with agitation. Blots as incubated in the secondary antibody (goat anti-rabbit DyLight® 800 conjugated, AS12 2460, Agrisera) 1:5000 dilution for 30min at RT with agitation and washed 1X with TBSTT for 15min, 1X with TBST for 15min before scanning with the ODyssey IRD scanner.
Courtesy of Dr. Betty Chung and Pawel Baster, University of Cambridge, United Kingdom
There can be a reaction with NEB broad range prestained protein ladder depending upon secondary antibody used.
AGO6 accumulates to lower levels in the nrpd1a mutant (Havecker at el. 2010). In addition, a non-specific band migrates to a similar location and thus protein was run on a 6% denaturing gel to obtain good separation.
If other secondary antibody is used than in the figure below, cross-reacting pattern can be different.
AGO6 (Argonaute 6) belongs to a group of argonaute proteins which are catalytic component of the RNA-incudes silencing complex (RISC). This protein complex is responsible for the gene silencing (RNAi).
Havecker el al. (2010). The RNA-directed DNA methylation Arabidopsis Argonautes functionally diverge based on expression and interaction with target loci. Plant Cell. 2010 Feb;22(2):321-34. doi: 10.1105/tpc.109.072199.
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