AUX1 | Auxin transporter protein 1 (rabbit antibody)
AS16 3159 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana
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Arabidopsis thaliana AUX1 was expressed in insect cells using baculovirus infection. Expression was driven by the strong polyhedrin promoter. Cultures of Sf9 cells (20 ml, approx. 2 x 107 cells) were infected with virus at multiplicities of infection (MOI) of 0.1, 1 and 2.5. 2 mL samples were harvested by centrifugation after 3 days, lysed (20 mM Tris/HCl, pH 7.4, 200 mM NaCl, 1 mM EDTA, 1% Tween 20, protease inhibitors and DNAseI) at 4°C for 30 min, sonicated (3 x 5 s pulses) and centrifuged. Cleared supernatant samples (whole cell lysates) were run on SDS-PAGE using 8 – 13% acrylamide gradient gels. After transfer to PVDF, the membrane was blocked with TBS-Tween with 10% milk powder overnight. Primary antibodies were applied at 1:5000 dilution in TBS-Tween with 5% milk powder for one hour at room temperature, washed x3 in TBS-Tween for 10 mins each and secondary antibodies (e.g. AS09 605 rabbit anti-goat HRP conjugated, Agrisera) applied diluted 1:10 000 as above. After washing as above, development was by ECL using chemiluminescent detection reagent, for 2 mins and the image captured by ImageQuant. As negative controls, samples of non-infected cells (NC) were run alongside the AUX1 extracts, as well as protein size markers (Mr).
The complete fusion protein (enhanced GFP-AtAUX1) was detected at close to 60 kDa A breakdown or truncated band was also seen at a little over 40 kDa.
Courtesty of Prof Richard Napier, University of Warwick, UK
Alternative names: AUX1, AUXIN RESISTANT 1, WAV5, WAVY ROOTS 5, PIR1, MAP1, MODIFIER OF ARF7/NPH4 PHENOTYPES 1, Auxin influx carrier protein 1, Polar auxin transport inhibitor-resistant protein 1.
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