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ATG8 | Autophagy-related protein

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

ATG8 | Autophagy-related protein in the group Antibodies for Plant/Algal  / Protein Modifications / Autophagy-related and Ubiquitin-like Proteins at Agrisera AB (Antibodies for research) (AS14 2769)

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How to cite this product:
Product name, number (Agrisera, Sweden)

Data sheet Product citations Protocols Customer reviews

Product Information

Immunogen

Fragment of recombinant ATG8 from Chlamydomonas reinhardtii, UniProt: A8JB85, 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: Q8S925

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 tubes briefly prior to opening them to avoid any losses that might occur from lyophilized material adhering to the cap or sides of the tubes.
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, Populus trichocarpa, Solanum lycopersicum
Predicted reactivity

Brassica napus, Micromonas sp., Physcomitrella patens, Pinus sitchensis, Solanum tuberosum, Volvox carteri


Species of your interest not listed? Contact us
Not reactive in No confirmed exceptions from predicted reactivity are currently known.

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

Additional information

Additional information

This product can be sold containing ProClin if requested.

This antibody is recognizing 1 ng of recombinant CrATG8

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.

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Background

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 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.
Upadhyaya and Jagadeeshwar Rao (2019). Reciprocal regulation of photosynthesis and mitochondrial respiration by TOR kinase in Chlamydomonas reinhardtii. Plant Direct Volume 3, Issue 11.
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.
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

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