AGO2 | Argonaute 2
AS13 2682 | Clonality: Polyclonal | Host: Rabbit | Reactivity: A. thaliana
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Capsella rubella, Solanum tuberosum
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Chlamydomonas reinhardtii, Nicotiana benthamiana, Solanum lycopersicum, Zea mays
300 µg/well of Arabidopsis thaliana protein from wilde type and AGO1-36 knock out, AGO1 knockdown mutant (1-25) were extracted by TCA-acetone precipitation (check protocol tab) from floral tissue and saturated in 8M urea were separated on 15% SDS-PAGE (1 mm thick gel) and blotted for 1hour to 0.2 µm nitrocellulose at 100V using wet transfer system. Blots were blocked with 0.5% cold fish gelatin (buffered in TBS) for 1hr at room temp with agitation. Blot was incubated in the primary antibody at a dilution of 1:250 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 30 min. at RT with agitation and washed 1X with TBSTT for 15min, 1X with TBST for 15min before scanning with the ODyssey IRD scanner.
AGO2 is enriched in AGO1 knockdown mutant, which agrees with already published data Harvey et al. (2011), PLOS One.
Courtesy of Dr. Betty Chung, University of Cambridge, United Kingdom
AGO2 protein is strongly induced by stress.
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.
Antibody incubation should be done over night in 4°C. Use of material with enriched AGO2 levels is recommened.
AGO2 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). AGO2 is probably involved in antiviral RNA silencing.
Dalmadi et al. (2019). AGO-unbound cytosolic pool of mature miRNAs in plant cells reveals a novel regulatory step at AGO1 loading. Nucleic Acids Res. 2019 Aug 8. pii: gkz690. doi: 10.1093/nar/gkz690.
You et al. (2019). FIERY1 promotes microRNA accumulation by suppressing rRNA-derived small interfering RNAs in Arabidopsis. Nat Commun. 2019 Sep 27;10(1):4424. doi: 10.1038/s41467-019-12379-z. (immunoprecipiation)
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