Anti-ATG4 | Autophagy protein 4 isoform (plant)

20-50 µg/well of total protein extracted freshly from true leaves of 4-week-old A. thaliana plants. Used backgrounds were wild type Col-0, atg4a-2/b-2 (Zou et al, 2025.Nat Comms. https://doi.org/10.1038/) and wild-type over-expressing tagged ATG4B (recently established in my group, not published).
10 ug/well of total protein extracted from whole cells of Chlamydomonas reinhardtii at the log phase of growth. Wild-type and atg4KO lines were used for protein extraction (Zou et al, 2025.Nat Comms. https://doi.org/10.1038/)
20 ug/well of total protein freshly extracted from leafy tissue of Physcomitrium patens of the wild-type and atg4a/b knockout backgrounds (recently established in my group, not published). Plant material was flash frozen in liquid N2, powdered and mixed 1:1 with 2xLaemmli buffer. Samples were boiled at 90C for 5 minutes, spund down at 17000g for 5 minutes-> supernatants were stored at -20C at preheated at 95C for 1 minute before loading on the gel Samples were separated in the cold on precast TGX, StainFree Bio-Rad 4-20% SDS-PAGE  and blotted for 7.5 minutes to PVDF using Bio-Rad premade cassettes and Turbo program for semi-dry transfer. Blot was blocked with 5% milk for 15 minutes at RT with agitation. Blot was incubated in the primary antibody at a dilution of 1:500 for 48h at 4°C. The antibody solution was decanted, and the blot was washed four times for 10 minutes in PBS-T at RT with agitation. Blot was incubated in a-rabbit HRP secondary antibody (AS09 602-trial Goat anti-rabbit HRP conjugated) diluted to 1: 50 000 in 2.5% skim milk for 1 h/RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagent: ECL, Bio-Rad. Exposure time was collected in accumulation mode, every second for the period of 100 seconds. After luminescence detection, the membrane was stained with Ponceau S for loading control.

Note: Shorter incubation time with ATG4 antibody will decrease background singal.

Courtesy of Dr. Dr. Alyona Minina, Swedish University of Agricultural Sciences, Sweden