Anti-AARE | Acyloamino acid releasing enzyme

Samples:
Arabidopsis thaliana (1-4) expressing an AtAARE:eGFP fusion (~110 kDa) under the control of the AtUbiq10 promoter (https://doi.org/10.1111/j.1365-313X.2010.04322.x) in the AtAARE mutant background (s68, https://doi.org/10.1038/s42003-023-04428-7).

Proteins were extracted from rosette leaves (~5–6 weeks old) in 50 mM PBS, 0.1% Triton X-100, 0.2 mM DTT, and 2 mM EDTA. For each sample, 50 μg of protein was mixed with Laemmli buffer containing β- mercaptoethanol, heated at 70°C for 5 minutes, and separated via SDS-PAGE. Proteins were transferred to a PVDF membrane (Merck, IPVH00010), and free binding sites were blocked with 5% milk (Roth, T145.3) overnight at 4°C with gentle agitation. The primary antibody (anti-AtAARE, AS23 4906) was diluted 1:1000 in TBS-T containing 2.5% milk (Roth, T145.3) and incubated with the membrane for 2 hours at room temperature with gentle agitation. The membrane was washed briefly twice, then twice for 5 minutes each, and once for 15 minutes in 30 mL TBS-T. The secondary antibody (anti-rabbit, HRP-conjugated secondary antibodies) was diluted 1:3000 in TBS-T with 2.5% milk (Roth, T145.3) and incubated with the membrane for 1 hour at room temperature with gentle agitation. Washing steps were repeated as described above. Detection was performed using chemiluminescent detection reagent and imaged using an ImageQuant 800 system (Cytiva).

Courtesy of Dr. Sebastian Hoernstein, University of Freiburg, Germany