AS10 673 Anti-AGO9 | Argonaute 9

80 µg of Arabidopsis thaliana soluble total cell extract (extracted in 20 mMTris pH7.5, 5mM MgCl2, 2.5mM DTT, 300mM NaCl, 0.1% NP-40, 1% proteaseinhibitor) 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-AGO9 antibody (1:10 000, 1h) and secondary anti-rabbit antibody (HRP conjugated, Agrisera AS09 602) (1:15 000, 1 h) in TBS-TT containing 5% low fat milk powder. Antibody incubations were followed by washings in TBS-TT. All steps were performed at RT with agitation. Blots were developed for 5 min with chemiluminescent detection reagent according the manufacturer's instructions. Exposure time was 30 seconds.
 

Courtesy Dr. Ericka Havecker, University of Cambridge, UK

AGO expression may be cell/tissue specific and using floral tissue is recommended where most of the AGOs are expressed the highest. Seedlings can be used as a negative control.

Use of proteasome inhibitors as MG132 can help to stabilize AGO proteins during extraction procedure.

A recommended whole-mount immunolocalization protocol can be found here. 

AGO9 belongs to a group of argonaute proteins which are catalytic component of the RNA-incudes silencing complex (RISC).

1. Hou et al. (2021) High-throughput single-cell transcriptomics reveals the female germline differentiation trajectory in Arabidopsis thaliana. Commun Biol. 2021 Oct 1;4(1):1149. doi: 10.1038/s42003-021-02676-z. PMID: 34599277; PMCID: PMC8486858. (immunolocalization)
2. Oliver & Martinez. (2021) Accumulation dynamics of ARGONAUTE proteins during meiosis in Arabidopsis. Plant Reprod. 2021 Nov 23. doi: 10.1007/s00497-021-00434-z. Epub ahead of print. PMID: 34812935.
3. Sprunck et al. (2019). Elucidating small RNA pathways in Arabidopsis thaliana egg cells. http://dx.doi.org/10.1101/525956
4. Su et al. (2017). The THO Complex Non-Cell-Autonomously Represses Female Germline Specification through the TAS3-ARF3 Module. Curr Biol. 2017 Jun 5;27(11):1597-1609.e2. doi: 10.1016/j.cub.2017.05.021.
5. 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.

TCA/Acetone protein precipitation method for plant proteins

• Grind plant tissue in a liquid nitrogen. 
• Continue grinding with 10% TCA solution in acetone (ice cold).
• Precipitate overnight in -20C.
• Spin at 4°C for 1min, 17k rpm > wash with ice cold acetone until you obtain a white pellet.
• Dissolve the pellet in buffer of choice (for example 8M urea containing 5mM DTT, or denaturate in SDS protein loading buffer for 10 min. at 70°C)
• Clarify supernatant
• Measure protein concentration. 
• Proceed with a western blot.

An example of a western blot result obtained with this method, which allows high protein load per well, can be found below. 

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 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 30 min. at RT with agitation and washed 1X with TBSTT for 15 min, 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

Recommended protocol for whole-mount immunolocalization using anti-AGO9 antibodies can be found here. 

Agrisera Western Blot protocol and video tutorials

Protocols to work with plant and algal protein extracts

Agrisera Educational Posters Collection