PR-1 | Pathogenesis-related protein 1
AS10 687 | clonality: polyclonal | host: rabbit | reactivity: Arabidopsis thaliana
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|Recommended dilution||1: 2500 (WB)|
|Expected | apparent MW||
17.7 kDa (Arabidopsis thaliana)
Arabidopsis thaliana, Hordeum vulgare, Zea mays, Spinacia oleracea, Solanum lycopersicum, Triticum aestivum
Brassica rapa subsp. pekinensis, Brassica napus, Eutrema japonica, Glycine max, Solanum tuberosum
|Not reactive in||
No confirmed exceptions from predicted reactivity are currently known
Re-using of antibody solution is not recommended. It will contribute to incrteased background signal.
Zhu et al. (2016). CML8, an Arabidopsis calmodulin-like protein plays a role in Pseudomonas syringae plant immunity. Plant Cell Physiol. 2016 Nov 10. pii: pcw189. [Epub ahead of print]
Western Blot -1
Recombinant PR-1 protein (0.2 pmol) (7) and 20 µg of a total protein from Arabidopsis thaliana (1), Hordeum vulgare (2), Zea mays (3), Spinacia oleracea (4), Solanum esculentum (5), Triticum aestivum (6) were extracted with Protein Extraction Buffer PEB (AS08 300), recombinant PR-1 (7) and were supplemented with 50 mM DTT and heat at 70°C for 5 min and kept on ice before loading. Protein separation was done using NuPage 4-12% Tris-Bis gel (Invitrogen) LDS-PAGE and blotted 1h to PVDF. Blots were blocked immediately following transfer in 2-2.5 % RPN2125 (GE Healthcare) in 20 mM Tris, 137 mM sodium chloride pH 7.6 with 0.1% (v/v) Tween-20 (TBS-T) for 1h at room temperature with agitation. Blots were incubated in the primary antibody at a dilution of 1: 2 500 in blocking reagent for 1h at room temperature 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 room temperature with agitation. Blots were incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, recommended secondary antibody AS09 602) diluted to 1:15 000 in TBS-T for 1h at room temperature with agitation. The blots were washed as above and developed for 5 min with TMA-6 (Lumigen) detection reagent according the manufacturers instructions. Images of the blots were obtained using a CCD imager (FluorSMax, Bio-Rad) and Quantity One software (Bio-Rad). Exposure time was 1 minute.
Western Blot -2
10 µg of total protein from Arabidopsis thaliana columbia strain from various experimental set ups, extracted with Sigma Aldrich Plant Total Extraction Kit were separated on 15 % SDS-PAGE and blotted 1h to PVDF. Blots were blocked with for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 500 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 0.1% at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from BioRad) diluted to 1:3 000 in for 1h at RT with agitation. The blot was washed as above and developed for 5 min with Biorad Opti-4Cn Substrate kit according to the manufacturer's instructions.
|Immunostaining of Arabidopsis thaliana seedlings treated with 250 µM SA for 120 minutes for induction of PR- gene expression (right panel) or control without SA (left panel). Steps involved: fixation: in 2 % formaldehyde in MTSB buffer for 30 minutes at 37 °C; washing with water, hydrophilization with methanol; cell wall digestion: 5-7 minutes, 0.25% Dricelaze, 0.1 % Macerozyme in 5 mM MES buffer, pH5.2; cell wall permeabilization: 10 % DMSO/3 % NP40 in MTSB buffer; primary antibody incubation: dilution 1: 200 in MTBS buffer for 3 h at RT; secondary antibody incubation: dilution 1: 1000 at RT for 1 h, goat-anti rabbit IgG Alexa 488 conjugated antibody.
Courtesy of Dr. Taras Pasternak, Freiburg University, Germany