AGO1 | Argonaute 1

AS09 527 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana, Nicotiana benthamiana

Product Information

Immunogen

KLH-conjugated, N-terminal peptide of Arabidopsis thaliana AGO1 O04379, At1g48410

Host Rabbit

Clonality Polyclonal

Purity Affinity purified serum in PBS pH 7.4.

Format Lyophilized

Quantity 50 µg

Reconstitution For reconstitution add 50 µl of sterile water.

Storage Store lyophilized/reconstituted at -20°C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please, remember to spin tubes briefly prior to opening them to avoid any losses that might occur from lyophilized material adhering to the cap or sides of the tubes.

Tested applications Chromatin ImmunoPrecipitation (ChIP), Immunolocalization (IL), small-RNA-IP-Seq, Western blot (WB)

Recommended dilution 2 µg (ChIP), 1: 200 (IL), small-RNA-IP-Seq, 1 : 5000-1 : 10 000 (WB)

Expected | apparent MW

116.4 | 130 kDa

Reactivity

Confirmed reactivity Arabidopsis thaliana, Nicotiana benthamiana

Predicted reactivity Brassica pekinensis, Capsella rubella, Glycine max, Malus domestica, Pisum sativum, Ricinus communis, Solanum tuberosum, Zea mays, Vitis vinifera

Not reactive in

Chlamydomonas reinhardtii, Triticum aestivum, Zea mays

Application examples

Application example

western blot using anti-AGO1 antibodies

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% proteasome 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) 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. Exposure time was 5 seconds.

Roche protease inhibitor cocktail (no EDTA) can also be applied in extraction buffer.

Courtesy Dr. Ericka Havecker, University of Cambridge, UK

Additional information

Antibody binds microRNA and tasiRNAs, preference for 21nt miRNAs with 5'U.

To detect AGO1 in Nicotiana benthamiana, please inquire.

Recommended for detection of AGO1: extreme low femtogram range chemiluminescent detection reagent

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. Buffer for extraction of AGO proteins: Paudel et al. 2018.

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.

TCA acetone precipitation method

Related products

Collection of antibodies to micro RNA

Plant protein extraction buffer

Secondary antibodies

Background

AGO1 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).

Product citations

Selected references 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
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.
Li (2019). The Isolation of Total and Membrane-Bound Polysomes from Arabidopsis and the Detection of Their Associated AGO1 and sRNAs. Methods Mol Biol. 2019;1932:317-333. doi: 10.1007/978-1-4939-9042-9_23.
Sprunck et al. (2019). Elucidating small RNA pathways in Arabidopsis thaliana egg cells. http://dx.doi.org/10.1101/525956
Chen et al. (2018). Structural and biochemical insights into small RNA 3' end trimming by Arabidopsis SDN1. Nat Commun. 2018 Sep 4;9(1):3585. doi: 10.1038/s41467-018-05942-7. (Immunoprecipitation)
Bologna et al. (2018). Nucleo-cytosolic Shuttling of ARGONAUTE1 Prompts a Revised Model of the Plant MicroRNA Pathway. Mol Cell. 2018 Feb 15;69(4):709-719.e5. doi: 10.1016/j.molcel.2018.01.007.
Zhang et al. (2017). RISC-interacting clearing 3'- 5' exoribonucleases (RICEs) degrade uridylated cleavage fragments to maintain functional RISC in Arabidopsis thaliana. Elife. 2017 May 2;6. pii: e24466. doi: 10.7554/eLife.24466.
Schalk et al. (2017). Small RNA-mediated repair of UV-induced DNA lesions by the DNA DAMAGE-BINDING PROTEIN 2 and ARGONAUTE 1. Proc Natl Acad Sci U S A. 2017 Mar 21. pii: 201618834. doi: 10.1073/pnas.1618834114.
Dolata et al. (2016). Salt Stress Reveals a New Role for ARGONAUTE1 in miRNA Biogenesis at the Transcriptional and Posttranscriptional Levels. Plant Physiol. 2016 Sep;172(1):297-312. doi: 10.1104/pp.16.00830. (ChIP and Immunolocalization)
Pumplin et al. (2016). DNA Methylation Influences the Expression of DICER-LIKE4 Isoforms, Which Encode Proteins of Alternative Localization and Function. The Plant Cell November 14, 2016 tpc.00554.2016 . Advance Publication November 14, 2016.
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.
Haveckeret al. (2010). The Arabidopsis RNA-directed DNA methylation argonautes functionally diverge based on their expression and interaction with target loci. Plant Cell. 2010 Feb;22(2):321-34. doi: 10.1105/tpc.109.072199. (small-RNA-IP-Seq)