NPR1 Nonexpresser of PR genes 1 (1 mg)

AS12 1854-1mg Clonality: Polyclonal Host: Rabbit | Reactivity: Arabidopsis thaliana


product information
Background		NPR1 (regulatory protein NPR1) is a key positive regulator of the SA-dependent signaling pathway that negatively regulates JA-dependent signaling pathway. Controls the onset of systemic acquired resistance (SAR). Subcellular localization: cytoplasm, nucleaus (following induction by salicylic acid treatment or after pathogen infection. Alternative names: BTB/POZ domain-containing protein NPR1, Non-inducible immunity protein 1, Nim1, Salicylic acid insensitive 1, Sai1, ATNPR1.
Immunogen		KLH-conjugated peptide, chosen from NPR1 sequence of Arabidopsis thaliana, TAIR: AT1G64280, UniProt: P93002
Host		Rabbit
Clonality		Polyclonal
Clone
Purity		Affinity purified serum in PBS, pH 7.4
Format		Lyophilized in PBS pH 7.4
Quantity		1 mg
Reconstitution		For reconstitution add 1 ml 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		AS10 687 \| anti-PR-1 \| Pathogenesis-related protein 1 AS09 412 \| anti-Salicylate \| 4-aminosalicylic acid Plant protein extraction buffer Secondary antibodies
Additional information		For successful detection using NPR1 antibody please follow protocol suggested below. NPR1 protein readily oligomerizes, in addition to any naturally occurring oligomer, during extraction. Therefore 50 mM DTT has to be used as well as denaturation at 75°C for 15 minutes. Engogenous NPR1 level is very low, thereofre SA treatment is absolutely necessary for good detection. This antibody is recognizing NPR1-GFP in the 35S overexpression line.

application information
Recommended dilution		1 : 1000 (WB)
Expected \| apparent MW		66 \| 66 kDa
Confirmed reactivity		Arabidopsis thaliana
Predicted reactivity		Arabidopsis thaliana
Not reactive in		Nicotiana benthamiana, Nicotiana tabacum, Solanum lycopersicum, Solanum tuberosum
Additional information		Please note that depending upon detection system you are using, longer exposure time may be required with this antibody.
Selected references		to be added when available, antibody released in December 2015.

application example

Samples (0.2 g) were collected from leaf tissue (3 week old rosettes; 24 hours after +/- 0.5 mM sodium salicylate spray). Total protein was extracted in a buffer containing 50 mM Tris-Hcl, pH 7.5; 150 mM NaC; 0.5 mM EDTA; 0.1 % Triton X-100; 0.2 % nonidet P-40; 50 uM MG115 and samples were adjusted to equal total protein concentration. Samples were denatured with 4X SDS Sample buffer with 200 mM DTT (final sample concentration of 50 mM) at 75 C for 15 min. Protein samples (30 µg of total protein) were separated on 4-12% Bis-Tris gel and blotted to PVDF using an iBlot (semi-dry transfer system; Life technologies). The membrane was blocked with 1X PBS-T containing 5% low fat dry milk ans 0.1 % tween-20, for 1h at room temperature (RT) with agitation and incubated in the primary antibody (in blocking solution) at a dilution of 1: 1 000 over night at 4 C with agitation. The antibody solution was decanted and the membrane was washed with blocking solution, 3 times for 10 min each at RT with agitation. The membrane was then incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:10 000 in blocking solution for 1h at RT with agitation. The membrane was washed as described above, rinsed with 1x PBS-T and developed for 2 min with Pierce West Dura extended duration substrate. Exposure time was 30 seconds.

** Note: 50 mM DTT concentration in the extraction buffer is necessary to reduce NPR1 oligomer formation.

** Note: The expression level of endogenous NPR1 is very low in extracts from healthy, untreated plants. A successful SA treatment or pathogen infection is absolutely necessary for good detection.

Courtesy Dr. John Withers, Duke University, USA

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