AS09 527-HRP Anti-AGO1 | Argonaute 1 (40 µg, HRP-conjugated)

Immunogen:	KLH-conjugated N-terminal peptide of Arabidopsis thaliana AGO1 UniProt: O04379, TAIR: At1g48410.
Host:	Rabbit
Clonality:	Polyclonal
Purity:	Immunogen affinity purified serum in PBS pH 7.4, conjugated to HRP.
Format:	Liquid
Quantity:	40 µg
Storage:	Store at 4°C for 12-18 months. A preservative may be added for long time storage up to 2 years.
Tested applications:	Western blot (WB)
Recommended dilution:	1 : 1000 (WB)
Expected | apparent MW:	116.4 | 130 kDa

Confirmed reactivity:	Arabidopsis thaliana, Hyacinthus orientalis, Nicotiana benthamiana
Predicted reactivity:	Brassica pekinensis, Capsella rubella, Glycine max, Malus domestica, Pisum sativum, Ricinus communis, Solanum tuberosum, Zea mays, Vitis vinifera Species of your interest not listed? Contact us
Not reactive in:	Chlamydomonas reinhardtii, Oryza sativa, Phaseolus vulgaris, Triticum aestivum, Zea mays

Additional information:

Antibody binds microRNA and tasiRNAs, preference for 21nt miRNAs with 5'U. TCA acetone total protein precipitation method

Additional information (application):

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.

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).

This antibody is directly conjugated to Horseradish Peroxidase (HRP).

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