ATG8 | Autophagy-related protein

AS14 2769 | Clonality: Polyclonal | Host: Rabbit | Reactivity: A. thaliana, A. madagascariensis, C. reinhardtii, H. lacustris, N. benthamiana, P. trichocarpa. S. lycopersicum

Product Information

Immunogen

Fragment of recombinant ATG8 from Chlamydomonas reinhardtii, UniProt: A8JB85

Host Rabbit

Clonality Polyclonal

Purity Serum

Format Lyophilized

Quantity 50 µl

Reconstitution For reconstitution add 50 µl of sterile water

Storage Store lyophilized/reconstituted at -20°C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please remember to spin the tubes briefly prior to opening them to avoid any losses that might occur from material adhering to the cap or sides of the tube.

Tested applications Immunolocalization (IL), Western blot (WB)

Recommended dilution 1 : 1000 (IL), 1 : 1000-1 : 2000 (WB)

Expected | apparent MW 15,2 | 15 kDa

Reactivity

Confirmed reactivity Arabidopsis thaliana, Aponogeton madagascariensis, Chlamydononas reinhardtii, Haematococcus lacustris, Nicotiana benthamiana, Populus trichocarpa, Solanum lycopersicum

Predicted reactivity

Ananas comosus, Brassica napus, Micromonas sp., Oryza sativa, Panicum hallii, Phoenix dactylifera, Physcomitrium patens, Pinus sitchensis, Solanum tuberosum, Volvox carteri

Species of your interest not listed? Contact us

Not reactive in Cuscuta chinensis

Application examples

Application examples Application example

western blot using anti-ATG8 antibodies on recombiant CrATG8

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

western blot using anti-CrATG8 antibodies

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

western blot with anti-ATG8 antibodies on Arabidopsis thaliana and Chlamydomonas reinhardtii samples

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: Chlamydomonas reinhardtii (Green Alga)

Application: Western Blotting

Pudmed ID: 32765574

Journal: Front Plant Sci

Figure Number: 6B

Published Date: 2020-08-09

First Author: Chen, X. D., Liu, Y., et al.

Impact Factor: 5.435

Open Publication

Effects of different H2S donors or H2S biosynthesis inhibitors on cell autophagy capability of C. reinhardtii under MC-LR treatment. (A) Effects of H2S biosynthesis scavenger or inhibitor on MC-LR-induced expression of ATG1, AGT7, AGT8, and ATG101. Here, 3-d-old C. reinhardtii cells were treated with 100 nM MC-LR with or without H2S artificial donor NaHS (100??M), GYY4137 (GYY, 10??M), H2S scavenger hypotaurine (HY, 10??M), or H2S biosynthesis enzyme inhibitor aminooxyacetic acid (AOA, 1?mM) for 12 h, with transcriptional levels of ATG1, AGT7, AGT8, and ATG101 then measured by qRT-PCR analysis. Values are means ± SD of three biological replicates. (B) Effects of H2S biosynthesis scavenger or inhibitor on MC-LR-induced protein accumulation of ATG8. Here, 3-d-old C. reinhardtii cells were treated with 100 nM MC-LR with or without H2S artificial donor for 3 d, with protein accumulation of ATG8 measured by Western blotting using anti-ATG8 antibody. Anti-Tubulin was used as the loading control.

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.

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.

Background

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).

Product citations

Selected references Zharova et al. (2022) Role of Autophagy in Haematococcus lacustris Cell Growth under Salinity. Plants. 2022; 11(2):197. https://doi.org/10.3390/plants11020197 (immunofluorescence)
Li et al. (2021) Chloroplast proteotoxic stress-induced autophagy is involved in the degradation of chloroplast proteins in Chlamydomonas reinhardtii. Plant Cell Physiol. 2021 Feb 17:pcab029. doi: 10.1093/pcp/pcab029. Epub ahead of print. PMID: 33594417.
Li et al. (2021) The unfolded protein response plays dual roles in rice stripe virus infection through fine-tuning the movement protein accumulation. PLoS Pathog. 2021 Mar 4;17(3):e1009370. doi: 10.1371/journal.ppat.1009370. Epub ahead of print. PMID: 33662041.
Sun et al. (2021) Mechanistic insights into an atypical interaction between ATG8 and SH3P2 in Arabidopsis thaliana. Autophagy. 2021 Oct 17:1-17. doi: 10.1080/15548627.2021.1976965. Epub ahead of print. PMID: 34657568.
Mishra et al. (2021) Interplay between abiotic (drought) and biotic (virus) stresses in tomato plants. Mol Plant Pathol. 2021 Dec 30. doi: 10.1111/mpp.13172. Epub ahead of print. PMID: 34970822.
De Brasi-Velasco et al. (2021). Autophagy Is Involved in the Viability of Overexpressing Thioredoxin o1 Tobacco BY-2 Cells under Oxidative Conditions. Antioxidants. 2021; 10(12):1884. https://doi.org/10.3390/antiox10121884
Kazibwe et al. (2020). TOR mediates the autophagy response to altered nucleotide homeostasis in a ribonuclease mutant. J Exp Bot. 2020 Sep 9;eraa410.doi: 10.1093/jxb/eraa410.
Shull et al. (2019). Anatase TiO2 nanoparticles induce autophagy and chloroplast degradation in thale cress (Arabidopsis thaliana). Environ Sci Technol. 2019 Jul 29. doi: 10.1021/acs.est.9b01648.
Upadhyaya and Jagadeeshwar Rao (2019). Reciprocal regulation of photosynthesis and mitochondrial respiration by TOR kinase in Chlamydomonas reinhardtii. Plant Direct Volume 3, Issue 11.
Wojciechowska et al. (2018). Autophagy counteracts instantaneous cell death during seasonal senescence of the fine roots and leaves in Populus trichocarpa. BMC Plant Biol. 2018 Oct 29;18(1):260. doi: 10.1186/s12870-018-1439-6. (immunolocalization)
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

Related products: ATG8 | Autophagy-related protein

Agrisera ECL kit (Bright/SuperBright)

AS16 ECL-S-N | low pico to mid femtogram and extreme low femtogram detection

This product can b...

From 26 €

Info

Add to cart

ATG8A-I | Autophagy-related protein 8A-I isoforms

AS14 2811 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana

224 €

Goat anti-Rabbit IgG (H&L), ALP conjugated

AS09 607 | Clonality: Polyclonal Host: Goat Reactivity: Rabbit IgG (H&L)

206 €

Goat anti-Rabbit IgG (H&L), HRP conjugated

AS09 602 | Clonality: Polyclonal | Host: Goat | Reactivity: Rabbit IgG (H&L)

201 €