AS14 2769 Anti-ATG8 | Autophagy-related protein

Confirmed reactivity:	Arabidopsis thaliana, Aponogeton madagascariensis, Chlamydononas reinhardtii, Chlorococcum dorsiventrale, Haematococcus lacustris, Nicotiana benthamiana, Populus trichocarpa, Solanum lycopersicum, Zea mays
Predicted reactivity:	Ananas comosus, Brassica napus, Hordeum vulgare,  Micromonas sp., Nelumbo nucifera, Oryza sativa, Panicum hallii, Parachlorella kessleri, Phoenix dactylifera, Pyrus x bretschneideri, Physcomitrium patens, Pinus sitchensis, Solanum tuberosum, Triticum aestivum, Volvox carteri Species of your interest not listed? Contact us
Not reactive in:	Cuscuta chinensis, Symbiodinium sp.

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

 

Reactant: Arabidopsis thaliana (Thale cress)

Application: Western Blotting

Pudmed ID: 36997511

Journal: Nat Commun

Figure Number: 2C

Published Date: 2023-03-30

First Author: Zeng, Y.

Impact Factor: 15.405

Open Publication

FREE1 interacts with ATG conjugation system and ESCRT-III under starvation conditions.a Transgenic Arabidopsis seedlings expressing YFP-FREE1 upon autophagy induction by carbon starvation for at least 18 h were subjected to GFP-Trap assay for IP-MS analysis. Identified FREE1-interacting partners were analyzed using STRING. b Immunofluorescence labeling of the endogenous ATG conjugation system proteins ATG5, ATG12b or ATG16 in Arabidopsis protoplasts transfected with GFP-FREE1 for 24 h before confocal laser scanning microscopy (CLSM) observation. White dash boxes indicate the 5 X enlarged areas. Scale bars, 10 μm. c GFP-Trap and co-IP analysis of GFP-FREE1 with endogenous ATG5, ATG12b, or ATG16 using native promoter driven GFP-FREE1 transgenic plants. Arabidopsis transgenic plants expressing GFP or GFP-FREE1 driven by the native promoter were subjected to protein extraction and IP with GFP-Trap, followed by western blot analysis with indicated antibodies. d Structural TEM analysis of high-pressure freezing/frozen substituted atg5-1 mutant plant root tips upon BTH treatment for 8 h. Black dash boxes indicated the 5 X zoom-in areas. Arrowheads indicated examples of the unsealed autophagosomal structures. Scale bars, 500 nm. e 5 X zoom-in areas from d. Arrowheads indicated examples of the unsealed autophagosomal structures. Scale bars, 500 nm. f Quantification analysis of the ratio of the unclosed autophagosomal structures (APs) in Col-0 and atg5-1 mutants per cross-cell section. Means±SD; n = 10 (Col-0) and n = 17 (atg5-1) cells, two-tailed unpaired t test; ***p < 0.001. All the imaging analysis and immunoblots were repeated at least for three times with similar results.

Additional information:

This product can be sold containing ProClin if requested. This antibody is recognizing 1 ng of recombinant CrATG8.  Antigen used to elicit this antibody is conserved from 70-80% in following ATG protein from Arabidopsis thaliana: ATG8a UniProt: Q8LEM4  ATG8B UniProt: Q9XEB5  ATG8c UniProt: Q8S927 ATG8d UniProt: Q9SL04, ATG8e UniProt: Q8S926 ATG8f UniProt: Q8VYK7 and conserved below 70% in: ATG8g UniProt: Q9LZZ9 ATG8h Uniprot: Q8S92 This antibody does not recognize all isoforms into the same degree.

Additional information (application):

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. 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.

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).

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Agrisera Western Blot protocol and video tutorials

Protocols to work with plant and algal protein extracts


Oxygenic photosynthesis poster by prof. Govindjee and Dr. Shevela

Z-scheme of photosynthetic electron transport by prof. Govindjee and Dr. Björn and Dr. Shevela