AGO4 | Argonaute 4 (1mg)
AS09 617-1mg | clonality: polyclonal | host: rabbit | reactivity: Arabidopsis thaliana, Hyacinthus orientalis
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|Recommended dilution||5 ĩg of antibody per 1 gram of a fresh tissue (IP), 1 : 100 (ICC), 1 : 4000 (WB)|
|Expected | apparent MW||
|Confirmed reactivity||Arabidopsis thaliana, Hyacinthus orientalis|
|Not reactive in||Hordeum vulgare, Solanum lycopersicum, Brassica oleracea|
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. Use a 6 % gel for protein separation, which is run longer to avoid a cross-reactivity at ca. 40 kDa.
Note that the AGO4 antibody reacts with the NEB prestained protein marker.
|Selected references||Zhai et al. (2015). A One Precursor One siRNA Model for Pol IV-Dependent siRNA Biogenesis. Cell. 2015 Oct 8;163(2):445-55. doi: 10.1016/j.cell.2015.09.032.
Han et al. (2014). SUVR2 is involved in transcriptional gene silencing by associating with SNF2-related chromatin-remodeling proteins in Arabidopsis. Cell Res. 2014 Nov 25. doi: 10.1038/cr.2014.156. (coimmunoprecipitation)
Zhang et al. (2013). DTF1 is a core component of RNA-directed DNA methylation and may assist in the recruitment of Pol IV. PNAS, May 14.
Havecker et al. (2010) The RNA-directed DNA methylation Arabidopsis Argonautes functionally diverge based on expression and interaction with target loci. Plant Cell 22(2): 321-34.
360 µg/well of Arabidopsis thaliana protein extracted by TCA-acetone precipitation from floral tissue 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 (DayLight 800) 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
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