ATG8 | Autophagy-related protein
AS14 2769 | Clonality: Polyclonal | Host: Rabbit | Reactivity: A. thaliana, A. madagascariensis, C. reinhardtii, S. lycopersicum
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1: 1000 (IL), 1:1000 - 1: 2000 (WB)
|Expected | apparent MW||
15.2 | 15 kDa
|Confirmed reactivity||Arabidopsis thaliana, Aponogeton madagascariensis, Chlamydononas reinhardtii, Solanum lycopersicum|
Brassica napus, Micromonas sp., Physcomitrella patens, Pinus sitchensis, Populus trichocarpa, Solanum tuberosum, Volvox carteri
|Not reactive in||
No confirmed exceptions from predicted reactivity known in the moment
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)
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.
|Selected references||Chen et al. (2016). The role of nitric oxide signalling in response to salt stress in Chlamydomonas reinhardtii. Planta. 2016 Sep;244(3):651-69. doi: 10.1007/s00425-016-2528-0. Epub 2016 Apr 26.
Gorovits et al. (2016). Tomato yellow leaf curl virus confronts host degradation by sheltering in small/midsized protein aggregates. Virus Res. 2016 Feb 2;213:304-13. doi: 10.1016/j.virusres.2015.11.020. Epub 2015 Dec 1
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