PDF1 | Plant defensin 1.1
AS16 3973 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Solanum lycopersicum
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KLH-conjugated synthetic peptide derived from Arabidopsis thaliana PDF1.1 UniProt: P30224-1, TAIR: At1g75830
The peptide sequence, it is perfectly conserved in following Arabidopsis thaliana isoforms: PDF1.2c, PDF1.2b, PDF1.2A, PDF1.3. PDF2 isoform sequence did not come in the blast.
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.
1 : 1000 (WB)
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Marker used is Prestained Protein SHARPMASS™ VI Protein MW marker (5-245 kDa) from Euroclone company.
sample 1:, 40 µg of Arabidopsis thaliana total protein from wildtype leaves 0 hour post infection with Botrytis cinerea (negative control);
sample 2: 40 µg of Arabidopsis thaliana total protein from mutant leaves (with induced expression of PDF1 mRNA) 0 hour post infection with Botrytis cinerea (positive control);
sample 3: 40 µg of Arabidopsis thaliana total protein from wild type leaves 24 hours post infection with Botrytis cinerea (positive control, PDF1 is expected to be induced by the pathogen used in the experiment);
sample 4: 40 µg of Arabidopsis thaliana total protein from mutant leaves (with induced expression of PDF1 mRNA) leaves 24 hours post infection with Botrytis cinerea (positive control);
samples "-", 10 µg of Arabidopsis thaliana total protein wild type seedlings grown in dark condition (Negative control)
Up to 40 µg/well of total protein extracted freshly from 6-week-old leaves of Arabidopsis thaliana with extraction buffer (125 mm Tris, pH 6.8, 4% [w/v] SDS, 20% [v/v] glycerol, 0.02% [w/v] bromophenol blue, 10% [v/v] β-mercaptoethanol) and denatured 95°C for 7 minutes were separated on a 4–20% Mini-PROTEAN® TGX™ Precast Protein Gels (biorad) SDS-PAGE and blotted 1h to PVDF (pore size of 0.2 µm), using Trans-Blot Turbo Transfer System (Bio-Rad). Blot was blocked with 5% milk for 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 for overnight (~16hours) with agitation in PBS-T with agitation at +4 °C. 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 Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:10 000 for 2h/RT with agitation. The blot was washed as above and developed for 3 min with Agrisera ECL SuperBright with ChemiDoc Imaging Systems (Bio-Rad). Exposure time was 10 seconds.
Left membrane: Western blot detection. Right membrane: staining.
Courtesy Dr. Ricardo Lorrani, University of Rome Sapienza, Italy
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