AS10 672 Anti-AGO6 | Argonaute 6

Confirmed reactivity:	Arabidopsis thaliana
Predicted reactivity:	Arabidopsis thaliana
Not reactive in:	Zea mays

360 µg/well of Arabidopsis thaliana protein extracted from leaf using TCA-protein precipitation (for method check protocol tab) 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 (goat anti-rabbit DyLight® 800 conjugated, AS12 2460, Agrisera) 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

Additional information:

There can be a reaction with NEB broad range prestained protein ladder depending upon secondary antibody used. AGO6 accumulates to lower levels in the nrpd1a mutant (Havecker at el. 2010). In addition, a non-specific band migrates to a similar location and thus protein was run on a 6% denaturing gel to obtain good separation. If other secondary antibody is used than in the figure below, cross-reacting pattern can be different.

Additional information (application):

AGO6 expression is cell-specific. Seedlings can be used as a negative control

AGO6 (Argonaute 6) 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).

1. 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.
2. Sprunck et al. (2019). Elucidating small RNA pathways in Arabidopsis thaliana egg cells. http://dx.doi.org/10.1101/525956
3. Havecker el al. (2010). The RNA-directed DNA methylation Arabidopsis Argonautes functionally diverge based on expression and interaction with target loci. Plant Cell. 2010 Feb;22(2):321-34. doi: 10.1105/tpc.109.072199.

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.

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

 

Agrisera Western Blot protocol and video tutorials

Protocols to work with plant and algal protein extracts

Agrisera Educational Posters Collection