AGO1 | Argonaute 1 (100 ĩg)
AS09 527-100 | clonality: polyclonal | host: rabbit | reactivity: Arabidopsis thaliana, Nicotiana benthamiana
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1: 5000 - 1: 10 000 (ECL Plus)
|Expected | apparent MW||
116.4 | 130 kDa
|Confirmed reactivity||Arabidopsis thaliana, Nicotiana benthamiana
Pisum sativum, Ricinus communis, Vitis vinifera, Capsella rubella, Brassica pekinensis
|Not reactive in||
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.
The AGO1 antibody is extremely specific to AGO1 and does not cross-react with other antibodies. The evidence is 1) the peptide to which it was raised is at the very N-terminus of the protein and is not present in other AGOs 2) aAGO1 does not cross react with the AGOs which are overexpressed (AGO2, AGO3, AGO4, AGO5, AGO6, AGO9) using a western blot.
|Selected references||Minoia et al. (2014). Specific Argonautes Selectively Bind Small RNAs Derived from Potato Spindle Tuber Viroid and Attenuate Viroid Accumulation In Vivo. J Virol. 2014 Oct 15;88(20):11933-45. doi: 10.1128/JVI.01404-14. Epub 2014 Aug 6.|
80 µg of Arabidopsis thaliana soluble total cell extract (extracted in 20 mMTris pH 7.5, 5mM MgCl2, 2.5mM DTT, 300 mM NaCl, 0.1% NP-40, 1% protease inhibitor MG132) was separated on 6% SDS-PAGE and blotted 1h to nitrocellulose. Filters were blocked 1h with 5% low-fat milk powder in TBS-TT (0.25% TWEEN20; 0.1% Triton-X) and probed with anti-AGO1 antibody (1:10 000, 1h) and secondary anti-rabbit (1:10000, 1 h) antibody (HRP conjugated, Santa Cruz(sc-2054)) in TBS-TT containing 5% low fat milk powder. Antibody incubationswere followed by washings in TBS-TT. All steps were performed at RT withagitation. Blots were developed for 5 min with ECL-PLUS detection reagent according the manufacturer's instructions (GE Healthcare). Exposure time was 5 seconds.
Courtesy Dr. Ericka Havecker, University of Cambridge
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