Pex14p | peroxisomal marker
AS08 372 | clonality: polyclonal | host: rabbit | reactivity: Arabidopsis thaliana
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|Recommended dilution||1: 10 000 with standard ECL (WB)|
|Expected | apparent MW||
55.5 | 75-65 kDa (depending on the gel system)
|Predicted reactivity||Arabidopsis thaliana|
|Not reactive in||no confirmed exceptions from predicted reactivity known in the moment|
|Additional information||Antibodies will detect Pex14 protein in both light- and dark-grown seedlings grown on petri dishes and in rosette leaves of adult plants grown in soil.|
|Selected references||Li et al. (2015). A Chaperone Function of NO CATALASE ACTIVITY1 Is Required to Maintain Catalase Activity and for Multiple Stress Responses in Arabidopsis. Plant Cell. 2015 Feb 19. pii: tpc.114.135095.
Woodward et al. (2014). A viable Arabidopsis pex13 missense allele confers severe peroxisomal defects and decreases PEX5 association with peroxisomes. Plant Mol Biol. 2014 Jul 10.
McMichael et al. (2013). Mediation of Clathrin-Dependent Trafficking during Cytokinesis and Cell Expansion by Arabidopsis STOMATAL CYTOKINESIS DEFECTIVE Proteins. Plant Cell, Oct. 31.
Farmer et al. (2013).Disrupting Autophagy Restores Peroxisome Function to an Arabidopsis lon2 Mutant and Reveals a Role for the LON2 Protease in Peroxisomal Matrix Protein Degradation. Plant Cell, Oct 31.
Twenty 5-day-old light grown Arabidopsis thaliana seedlings (wild type, the pex14-2 null allele, and the pex14-1 truncation allele) were ground with a pestle in a 1.5 mL tube on dry ice following the addition of a double volume (60 μL) of NuPAGE 2x loading buffer (Invitrogen). After centrifugation, 20 μL of supernatant was trasnferred to a fresh tube with 2.1 μL 0.5 M DTT and boiled at 100 °C for 5 mins. Samples were loaded onto NuPAGE 10% Bis-Tris gel (Invitrogen) next to Cruz Marker (Santa Cruz Biotechnology). After electrophoresis, proteins were transferred for 30 mins at 24 V to a Hybond ECL nitrocellulose membrane (Anersham Pharmacia Biotech) using NuPAGE transfer buffer (Invitrogen). The blot was blocked for 1 h 4 °C in 8% non-fat dry milk in TBS-T (blocking buffer),and incubated overnight wiht agitation at 4 °C with primary antibody (1:10 000 rabbit anti-PEX14, AS08 372, Agrisera) diluted in blocking buffer. The antibody soliution was decanted and the blot was rinsed 3 times, 5 min each, with blocking buffer at 4 °C. The blot was incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:5000, Santa Cruz Biotechnology), diluted in blocking buffer for 4 h at 4 °C with agitation. The blot was washed with 3 times, 5 min each, with TBS-T and developed with Western Bright reagent (Advanta), 1:10 dilution in water.
Courtesy Sarah Burkhart and Bonnie Bartel, Rice University, USA.
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