ATG8 | Autophagy-related protein

Anti-CrATG8 antibodies detect 1 ng of recombinant CrATG8 protein.

30 µg of total protein from Chlamydomonas reinhardtii , control (C), autophagy induced (A), extracted with lysis buffer according to Perez-Perez et al. 2010 (Plant Physiology 152: 1874-1888) were separated on 15 % SDS-PAGE and blotted 1h to nitrocellulose membrane using semi-dry or tank transfer. Blots were blocked with 5% milk for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1:1000 for 1h at RT 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, from Agrisera AS09 602, diluted to 1:25 000) 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 45 seconds.

Courtesy of Dr. María Esther Pérez-Perez, IBVF, Spain

15 µg of total protein from Chlamydomonas reinhardtii and Arabidopsis thaliana were separated on 15 % 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 1:1 000 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 from Agrisera) diluted to 1:10 000 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 60 seconds.

Courtesy of Dr. María Esther Pérez-Pérez and Ana M. Laureano-Marín, IBVF, Spain

For Arabidopsis thaliana the signal obtained using ATG8 antibodies is cleaner in case of roots compare to leaf material. For best results please follow extraction protocol described in Álvarez et al. (2012). ATG8 signal corresponds to the two bands of 17 kDa.

Preparation of a cell extract from Arabidopsis thaliana:

A. Plants were first subjected to autophagy activating conditions: nutrient (nitrogen or carbon) limitation or oxidative stress in order to activate this degradative process.

B. Total protein extracts can be obtained as described by Álvarez. Leaves are grinded in liquid nitrogen with a minimal volume of extraction buffer (100 mM Tris-HCl pH 8, 400 mM sucrose, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mg/ml sodium deoxycholate, 10 µg/ml of leupeptin, 10 µg/ml of pepstatin A, 4% (v/v) protease inhibitor cocktail from Roche).

C. Cell debris is removed by centrifuging at 500 g for 10 min at 4°C.

It is recommendable to use bigger gels in order to get a better resolution of ATG8 bands. Midi-protean gels are better than mini-gels. There are 9 ATG8 isoforms and this antibody will likely recognizes all of them.

For immunolocalization protocol, please inquire.

ATG8 (Autophagy-related protein 8) is involved in degradation and recycling of intracellular components in a process of autophagy. ATG8 is a molecular autophagy marker in Chlamydomonas reinhardtii (Pérez-Pérez et al. 2010, Plant Physiol. 152: 1874-88).