AUX1 | Auxin transporter protein 1 (goat antibody)
AS16 3957 | Clonality: Polyclonal | Host: Goat | Reactivity: Arabidopsis thaliana
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Store lyophilized/reconstituted at -20°C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles.
Please, remember to spin tubes briefly prior to opening them to avoid any losses that might occur from lyophilized material adhering to the cap or sides of the tubes.
no confirmed exceptions from predicted reactivity known in the moment
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 Millipore Immobilon Transfer 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
Comparison between the two alleles: aux1-21 has a frame shift after exon 5, whereas aux1-2 only has an amino acid substitution and expression is checked in the protophloem.
Whole mount tissue immunochemistry of sections of protophloems of Arabidopsis thaliana root tips.
Fixation: 4% PFA in MTSB Buffer pH 6.9 Vacum 2x5’ and 1x40’
Blocking: BSA in MTSB (0.4g in 20ml), As a secondary antibody Alexafluor 546 donkey, anti-goat, concentration 1:500.
Courtesy Ana Cecilia Aliaga Fandino ,Hardtke lab, University of Lausanne, Switzerland
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