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ATG8 | Autophagy-related protein

265 €
Buy 2 items of this product for 198 €/each
Buy 3 items of this product for 180 €/each

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

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AS14 2769

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product information
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).
Immunogen

Fragment of recombinant ATG8 from Chlamydomonas reinhardtii, UniProt: A8JB85, conserved from 70-80 % in following ATG protein from Arabidopsis: 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
Clone
Purity Serum
Format Lyophilized
Quantity 50 µl
Reconstitution For reconstitution add 50 ĩl 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)
Related products

AS14 2811 | anti-ATG8A | Autophagy-related protein 8a, rabbit antibodies
AS14 2769PRE | ATG8 | Autophagy-related protein, pre-immune serum

Plant and algal protein extraction buffer

Secondary antibodies

Additional information

This product can be sold containing proClin if requested.

This antibody is recognizing 1 ng of recombinant CrATG8

application information
Recommended dilution 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
Predicted reactivity

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

Not reactive in No confirmed exceptions from predicted reactivity are currently known.
Additional information
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.

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.
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

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 Luminata Crescendo (Millipore) 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 Luminata Crescendo (Millipore) 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

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