Polyclonal AtEH1/Pan1 antibody generation

The Biotem antibody service (https://www.biotem-antibody.com/) was used to generate a polyclonal rabbit antibody against the C-terminus of AtEH1/Pan1. Rabbits for immunization were selected based on their blood serum responsiveness to the Arabidopsis proteome. Selected rabbits were injected on day 0, 7, 14 and 34 with purified protein concentrated to 0,724 mg/ml. After 42 days, the rabbits were exsanguinated and their sera were tested against the antigen. The antibody of the most responsive serum was purified by Biotem against the C-terminus of AtEH1/Pan1. The purification (Biotem) was performed using a second batch of recombinant protein concentrated at 3 mg/ml.

To test the purified antibody for specificity, 5-day-old seedlings expressing AtEH1/Pan1-mRuby3 in a Col-0 or ateh1/pan1 mutant background³⁵ as well as Col-0 control seedlings were shock frozen and ground in liquid nitrogen. Subsequently, heated 1.2x Laemmli buffer was added to the plant material followed by thorough vortexing. After 8 min incubation at 75 °C, samples were centrifuged two times at 20,000 g for 1 min at 4 °C. Samples were separated on a 4-20% Gradient TGX SDS stain free gel (BioRad) and comparable protein amounts between samples were confirmed via stain-free imaging (Extended Data Fig. 3d). The gel was blotted on a nitrocellulose membrane (BioRad) and developed with the purified polyclonal antibody diluted 1:2000 in PBS-T containing 5% milk powder. The specificity could be confirmed, as Col-0 and the AtEH1/Pan1-mRuby3 (Col-0) lines show the native AtEH1/Pan1 band roughly at 140 kDa, which is not present in the complemented mutant lines (Extended Data Fig. 3d, marked by black arrowheads). Transgenic lines expressing AtEH1/Pan1-mRuby3 in (Col-0) and (ateh1/pan1 1-2 -/-) backgrounds also exhibited a band for AtEH1/Pan1-mRuby3 at roughly 175 kDa (Extended Data Fig. 3d, marked by white arrowheads).