Fragment of recombinant ATG8 from Chlamydomonas reinhardtii, UniProt: A8JB85
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 (Perez-Perez et al. 2010, Plant Physiol. 152: 1874-88).
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 Alvarez 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 Alvarez. 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 4C.Important note: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.
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