AGO5 | Argonaute 5
AS10 671 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana
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111 | 111 kDa
Hordeum vulgare, Zea mays
Arabidopsis thaliana total protein extracted by TCA-acetone precipitation (check protocol tab for details) from floral buds of Col-0 (which should be more enriched in AGO5) was saturated in 8M urea were separated on 10% 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 a dilution of 1:250 overnight at 4C 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, Dr Pawel Baster, University of Cambridge, United Kingdom
AGO expression may be tissue specific and using floral tissue is recommended where most of the AGOs are expressed the highest. Use of proteasome inhibitors as MG132 can help to stabilize AGO proteins during extraction procedure.
AGO5 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). Probably involved in antiviral RNA silencing.
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