AGO10 | Argonaute 10
AS15 3071 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana
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A. lyrata, B. napus, C. rubella, C. clementina, C. sinensis, E. salsugineum, G. arboreum, G. raimondii. N. benthamiana
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50 µg of total protein from Arabidopsis thaliana inflorescences were extracted with extraction buffer (50 mM Tris pH7.5; 150 mM NaCl; 1 mM EDTA; 10 % v/v Glycerin; 1 mM DTT, 1x Complete Protease Inhibitor Cocktail, Roche) and denatured with Laemmli buffer at 95ºC 5 min. were separated on 10% SDS-PAGE and blotted 1.5 h to PVDF using tank transfer. Blots were blocked with blocking buffer (3% milk powder; 1x TBS; 0.1% Tween-20) 1 h at RT with agitation. Blot was incubated in the primary antibody at a dilution of 1:10 000 ON at 4ºC with agitation. The antibody solution was decanted and the blot was rinsed briefly and then washed tree times for 15 min. in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from Agrisera AS09 602) diluted to 1:20 000 in blocking buffer for 1h at RT with agitation. The blot was washed as above and developed for 5 min with chemiluminescent detection reagent of extreme femtogram sensitivity, exposed to Amersham Hyperfilms ECL for 5 minutes.
Courtesy of Dr. Dr. Pablo Manavella, Instituto de Agrobiotecnología del Litoral (IAL), Argentina
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.
AGO10 is involved in miRNA binding, and in RNA-mediated posttranscriptional gene silencing (PTGS).
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