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AGO1 | Argonaute 1 (100 ĩg)

AS09 527-100 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana, Nicotiana benthamiana

AGO1 | Argonaute 1 (100 ĩg) in the group Plant/Algal Antibodies / DNA/RNA/Cell Cycle / microRNA at Agrisera AB (Antibodies for research) (AS09 527-100)

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product information
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).

Immunogen

N-terminal peptide of Arabidopsis thaliana AGO1 O04379, At1g48410

Host Rabbit
Clonality Polyclonal
Clone
Purity Affinity purified serum in PBS pH 7.4
Format Lyophilized
Quantity 2 x 50 µg
Reconstitution For reconstitution add 50 µl of sterile water to each tube.
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 ImmunoPrecipiation (ChIP), Immunolocalization (IL), small-RNA-IP-Seq, Western blot (WB)
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collection of antibodies to micro RNA

Plant protein extraction buffer

Secondary antibodies

Additional information

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

This antibody is the same as AS09 527.

application information
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

Confirmed reactivity Arabidopsis thaliana, Nicotiana benthamiana
Predicted reactivity Brassica pekinensis, Capsella rubella, Malus domestica, Pisum sativum, Ricinus communis, Solanum tuberosum, Zea mays, Vitis vinifera
Not reactive in

Chlamydomonas reinhardtii, Triticum aestivum

Additional information

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.

TCA acetone precipitation method

Selected references 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)

application example

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



 

western blot using anti-AGO1 antibodies

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