ATG4 | Autophagy protein 4 (algal)

10 ng of recombinant, purified Chlamydomonas reinhardtii ATG4 (left) and 15 µg of Chlamydomonas reinhardtii total cell extract (right) were separated on 12 % SDS-PAGE and blotted 1h to nitrocellulose membrane using semi-dry transfer. Blots were blocked with 5 % dry milk in PBS for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1:5000 over night at 4 ºC with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, AS09 602, Agrisera) diluted to 1:10000 in 5 % dry milk for 1h at RT with agitation. The blot was washed as above and developed for 5 min with chemiluminescent detection reagent, according to the manufacturer's instructions. Exposure time was 30 seconds

Courtesy of María Esther Pérez-Pérez and Jose L. Crespo, CSIC, Spain

Technical details how to work with this antibody are provided here: Pérez-Pérez et al. (2016). Control of Autophagy in Chlamydomonas Is Mediated through Redox-Dependent Inactivation of the ATG4 Protease. Plant Physiol. 2016 Dec;172(4):2219-2234.

ATG4 (Autophagy protein 4) is a protein with cyteine-type endopeptidase activity involved in autophagy process.