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ACD1 | Accelerated cell death 1

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AS11 1783 | clonality: polyclonal | host: rabbit | reactivity:Arabidopsis thaliana

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Item No:
AS11 1783

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product information
Background

Acd1 (accelerated cell death 1) EC=1.14.12.20 is an enzyme involved in chlorophyll breakdown, pheophorbide a oxygenase. This enzyme seems to induce cell death in Arabidopsis leaves in the dark. Synonymes:PaO, Lls1, lethal leaf-spot 1 homolog, pheide a oxygenase

Immunogen

Recombinant PaO from Arabidopsis thaliana Q9FYC2, At3g44880

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 western blot (WB)
Related products

collection of antibodies to proteins involved in senescence

Plant protein extraction buffer

Secondary antibodies

Additional information

The protein level is moderately induced during dark-induced senescence.

application information
Recommended dilution

1 : 5000 with standard ECL (WB)

Expected | apparent MW

61 | 54 kDa

Confirmed reactivity Arabidopsis thaliana
Predicted reactivity dicots including: Brassica napus, Solanum lycopersicum, Nicotiana tabacum
Not reactive in

no confirmed exceptions from predicted reactivity are currently known

Additional information

This antibody works on total cell extracts and can be used as a senescence marker. Predicted size of Acd1 precursor protein is about 61 kD including the transit peptide, but it must be processed to a smaller size. Using fresh extracts is recommended to decrease possible cross-reaction with Rubisco.

Selected references Kim et al. (2013). Mutation of the Arabidopsis NAC016 Transcription Factor Delays Leaf Senescence.'Plant Cell Physiol. Aug 21.
Nagane et al. (2010). Involvement of AtNAP1 in thre reulation of chlorophyll degradation in Arabiopsis thaliana. Planta (4):939-949.
Hirashima et al. (2009). Light-independent cell death induced by accumulation of pheophorbide a in Arabidopsis thaliana. Plant Cell Physiol. (4):719-729.

application example

western blot using Acd1 antibody

Arabidopsis thaliana wild ecotype Columbia was grown for four weeks under continuous illumination and then transferred to complete darkness for five days. Several leaves were harvested from the plants before they were transferred to darkness (0 d) or after they were kept for five days (5 d). Protein was extracted with the SDS extraction solution containing 50 mM Tris (pH 6.8), 10% (w/v) glycerol, 2% (w/v) SDS and 6% (v/v) 2-mercaptoethanol. Protein extract equivalent to 1 mg leaf material was loaded and separated on 14% SDS-PAGE and blotted 1h to PVDF. Blots were blocked with PBS-T containing 1.5% skim milk for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 10 000 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 PBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from GE Healthcare ) diluted to 1:20 000 in for 1h at RT with agitation. The blot was washed as above and developed for 1 min with ECLplus according to the manufacturers instructions. Exposure time was 5 min.


western blot using anti-Acd1 antibodies

Arabidopsis thaliana wild ecotype Columbia was grown for four weeks under continuous illumination. Several young (1), mature (2) and senescing (3) leaves were harvested from the plants. Protein was extracted with the SDS extraction solution containing 50 mM Tris (pH 6.8), 10% (w/v) glycerol, 2% (w/v) SDS and 6% (v/v) 2-mercaptoethanol. Protein extract equivalent to 1 mg leaf material was loaded and separated on 14% SDS-PAGE and blotted 1h to PVDF. Blots were blocked with PBS-T containing 1.5% skim milk for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1:30 000 for 1h at RT with agitation as indicated in the figure. 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 PBS-T at RT with agitation. Blot was incubated in the secondary antibody provided by AgriSera (AS09 602) diluted to 1:20 000 in for 1h at RT with agitation. The blot was washed as above and developed for 1 min with ECLplus according to the manufacturers instructions. Exposure time was 5 min.

Courtesy of Kaori Takahashi at Hokkaido University, Japan

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