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 µl 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 Collection of antibodies to proteins involved in oxidative stress 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

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

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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