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RBOHD | Respiratory burst oxidase homolog protein D

265 €
Buy 2 items of this product for 198 €/each
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AS15 2962  | Clonality: polyclonal  |  Host: rabbit  |  Reactivity: Arabidopsis thaliana

PRODUCT INFORMATION IN PDF

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Item No:
AS15 2962

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product information
Background RBOHD (Respiratory burst oxidase homolog protein D) is a Calcium-dependent NADPH oxidase that generates superoxide. Is involved in the generation of reactive oxygen species (ROS) during incompatible interactions with pathogens and in UV-B and abscisic acid ROS-dependent signaling.
Immunogen

KLH-conjugated peptide chosen from Arabidopsis thaliana RBOHD sequence, UniProt:Q9FIJ0, TAIR: AT5G47910

Host Rabbit
Clonality Polyclonal
Clone
Purity Affinity purified serum
Format Lyophilized in PBS pH 7.4
Quantity 50 µg
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)
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Plant protein extraction buffer

Secondary antibodies

Additional information
application information
Recommended dilution

1 : 1000 (WB)

Expected | apparent MW

104 kDa

Confirmed reactivity Arabidopsis thaliana
Predicted reactivity Brassica napus, Citrus sinensis, Citrullus colocynthis, Fragaria ananassa, Gossypium arboreum, Manihot esculenta, Morus notabilis, Nicotiana tabacum, Oryza sativa, Picea abies, Ricinus communis, Solanum tuberosum, Theobroma cacao, Zea mays, Zostera marina
Not reactive in

 

Additional information RBOHD is a protein with 6 transmembrane-spanning regions, heat denaturation will cause protein aggregation. Therefore 30 minutes at RT was applied in the experiment below.
Selected references To be added when available, antibody released in May 2016.

Application example

western blot using anti-Arabidopsis RBOHD antibodies

40, 60 or 80 µg of total protein from Arabidopsis thaliana 10 days old seedlings,  extracted with 50 mM Tris-HCl (pH7.5), 2% SDS, 1:100 protease inhibitor cocktail  and denatured at 95°C 5 min. were separated on 7.5% SDS-PAGE  and blotted 1.5 h to PVDF using tank transfer. Blots were blocked with 5 % milk in TBS-T for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 for ON at 4°C with agitation. The antibody solution was washed 3 times for 10 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG IRDye800) diluted to 1:20 000 in Odyssey blocking buffer for 1h at RT with agitation. Blot was washed 3 times of 10 min in TBS-T at RT with agitation and rinsed with TBS.

Courtesy of Dr. Sachie Kimura and Dr. Michael Wrzaczek, Department of Biosciences, University of Helsinki, Finland


western blot using anti-RBOHD polyclonal antibodies

Non-transfection (1), 3xFLAG-AtRBOHD (2), 3xFLAG-AtRBOHF (3), 3xFLAG-AtRBOHC (4), atrbohD 25.6 µg (5), atrbohD/F 21.8 µg (6), atrbohF 20.11 µg (7): transient expression in HEK293T cells (extracted in a following way: Remove medium (HEK cell is adherent cell) 2. add 1xSDS buffer [62.5 mM Tris-HCl(pH6.8), 2% SDS, 5% sucrose, BPB, 10% mercaptoethanol, 300 mM DTT] 3. sonication 4. RT 30 min 5. SDS-PAGE);  Arabidopsis thaliana 10 days old seedlings Col-0 (8), 35S::FLAG-RBOHD, 6 weeks old Arabidopsis thaliana leaf (9), extracted with 50 mM Tris-HCl (pH7.5), 2% SDS, 1:100 protease inhibitor cocktail  and denatured at RT for 30 min. were separated on 10% SDS-PAGE and blotted 1.5 h to PVDF using tank transfer. Blots were blocked with 5 % milk in TBS-T for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 for 1h at RT with agitation. The antibody solution was washed 3 times for 10 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG IRDye800) diluted to 1:20 000 in Odyssey blocking buffer for 1h at RT with agitation. Blot was washed 3 times of 10 min in TBS-T at RT with agitation and rinsed with TBS.


Courtesy of Dr. Sachie Kimura and Dr. Michael Wrzaczek, Department of Biosciences, University of Helsinki, Finland

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