AGO1 | Argonaute 1 (Chlamydomonas)
AS14 2776 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Chlamydomonas reinhardii
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recombinant AGO1 of Chlamydomonas reinhardtii
Molecular weight markers (1), recombinant AGO1 of Chlamydomonas reinhardtii (2), recombinant AGO1 of Chlamydomonas reinhardtii (1/2 dilution) (3). Recombinant protein was purified using Ni-NTA Agarose (Qiagen), eluted in 50mM NaH2PO4, 500 mM NaCl and 250 mM imidazole (pH 7.5) and diluted in 1xPBS with 2% SDS. Samples were denatured prior to loading with 5 x loading buffer (350 mM Tris-Cl (pH 7.5), 30% glycerol, 10% SDS and 0.6 M DTT) by heating at 95°C for 5 mins. Samples were separated on NuPAGE 7% Tris-Acetate Protein Gel and blotted 1h to PVDF using tank transfer. Blots were blocked with 1xTBS with 0.1% Tween-20 (TBS-T) and 5% milk for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1000 for 15 hrs at 4°C with agitation in TBS-T. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (goat anti-rabbit Ab) diluted to 1:5000 in TBS-T for 1h at RT with agitation. The blot was washed as above and imaged using the Odyssey Infrared Imager and Image Studio v 2.1.
Courtesy of PhD candidate Claire Agius, University of Cambridge, UK
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).
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