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Toc75 | Protein TOC75-3, chloroplastic, POTRA domain 3
AS08 351 | Clonality: Polyclonal | Host: Rabbit | Reactivity: A.thaliana, N. benthamiana, N. tabacum, P. patens, P. sativum, S. oleracea, T. salsuginea, some cross-Reactivity was observed for cyanobacteria including: Synechocystis, Synechococcus and Thermosynechococcus sp.
compartment marker of chloroplast outer envelope membrane protein marker.
- Product Info
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Immunogen: Pisum sativum TOC75; Predicted POTRA Domain #3; Expressed and purified in E. coli using the Impact System from NEB. Peptide confirmed by MALDI. UniProt: Q43715
Host: Rabbit Clonality: Polyclonal Purity: Serum Format: Lyophilized Quantity: 50 µl 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 the tubes briefly prior to opening them to avoid any losses that might occur from material adhering to the cap or sides of the tube. Tested applications: Flow Cytometry (Flow cyt), Immunolocalization (IL), Western blot (WB) Recommended dilution: 1 : 500 (Flow cyt), 1 : 100 (IL), 1 : 2000-1 : 100 000 (WB) Expected | apparent MW: 88 | 75 kDa (ocassionally a processing intermediate at 78 kDa is observed)
- Reactivity
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Confirmed reactivity: Arabidopsis thaliana, Nicotiana benthamiana, Nicotiana tabacum, Physcomitrium patens, Pisum sativum, Spinacia oleracea, Thellungiella salsuginea, some cross-reactivity was observed for cyanobacteria including: Synechocystis, Synechococcus and Thermosynechococcus sp. Predicted reactivity: Aegilops tauschii, Ananas comosus, Anthurium amnicola, Capsicum annuum, Catalpa bungei, Cicer arietinum, Cucumis melo, Glycine soja, Gossypium arboreum, Medicago truncatula, Morus notabilis, Nelumbo nucifera, Nicotiana sylvestris, Noccaea caerulescens, Populus trichocarpa, Ricinus communis, Sorghum bicolor, Theobroma cacao, Zea mays, Zostera marina, Vigna radiata, Vitis vinifera
Species of your interest not listed? Contact usNot reactive in: No confirmed exceptions from predicted reactivity are currently known - Application Examples
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Application example
Samples of crude Arabidopsis thaliana chloroplast preparation from 250xg pellet (1), unknown sample (2), Arabidopsis thaliana 100kDa membrane preparation (3), prestained marker 80kDa band (NEB Broad Range) (4), Physcomitrella patens ‘broken’ chloroplasts from above 40% on Percoll gradient (5), unknown sample (6), Physcomitrella patens intact chloroplasts from above 80% on Percoll gradient (7) were blotted to PVDF membrane. Blot was incubated overnight at 4°C in 5% milk powder in Tris Buffered Saline (TBS), rinsed twice and then washed for 3 x 15 min in TBS+ 0.1%Tween20 (TBS-T). Incubation in anti-Toc75 POTRA3 antibody was done for 2h/RT using 8μl antibody per 10 ml 3% milk powder in TBS-T. Washes: 3 x 15 min in TBS+ 0.1%Tween20 (TBS-T). Incubation in goat anti-rabbit peroxidase secondary antibody was for 1h at room temperature and used 4μl antibody per 10ml 3% milk powder in TBS-T. Washes: 3 x 15 min in TBS+ 0.1%Tween20 (TBS-T) followed by incubation with chemiluminescence detection reagent and exposure for 10 min or 2min in an ImageQuant LAS4000 (lanes 1-3 and 5-7, respectively).
Courtesy Dr. Amanda Dowson, Warwick University, UK
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580 ng of Chl of Pisum sativum plants (10 day old) (2, 4) and 10 µg of combined envelopes of Pisum sativum 10 day-old (1,3) were separated on 15% SDS-PAGE and blotted 2h to PVDF. PVDF was blocked 1h with 3% non-fat milk powder in TBS-T (0.1% TWEEN 20) and probed with anti-Toc75 POTRA domain 3 antibodies AS08 351 (1:2000 and 1: 5000, 1h) and secondary donkey-anti-rabbit (1:20000, 1 h) antibody (HRP conjugated) in TBS-T containing 3% non fat milk powder. Antibody incubations were followed by washings in TBS-T. All steps were performed at RT with agitation. Blots were developed for 5 min with chemiluminescence detection reagent according to the manufacturers instructions. Exposure time was 600 seconds.
Ashita Dave and Barry D. Bruce (UTK), USA
- Additional Information
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Additional information (application): Antibody detects Toc75 POTRA domain 3 as purified protein, in chloroplast fraction and in crude envelope fraction - Background
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Background: OEP 75 or Toc75; Chloroplast outer envelope membrane protein from Pisum sativum (pea), Predicted to contain 3 POTRA domains at N-terminus. Believed to be the protein conducting channel of the Toc translocon and assembles as an 18 stranded ß-barrel (EMBO J. (1995) 14:11, 2436-2446); In Arabidopsis there are five members of this Family Toc75 (I-V), atToc75III is most closely related to psToc75. Additionally, it is structurally related to members of the bacterial surface antigen super-family including: OMA87; Outer membrane protein/protective antigen, (COG4775, COG4775) [Cell envelope biogenesis, outer membrane; YaeT; outer membrane protein assembly complex, (TIGR03303); FhaC; Hemolysin activation/secretion protein (COG2831) [Intracellular trafficking and secretion]
- Product Citations
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Selected references: Sasaki and Yamamoto (2015). Arabidopsis LAZY1 is a peripheral membrane protein of which the carboxy-terminal fragment potentially interacts with microtubules. Copyright © 2015 The Japanese Society for Plant Cell and Molecular Biology, Plant Biotechnology, 32, 1–6 (2015) DOI: 10.5511/plantbiotechnology.15.0116a
Vera-Estrella et al. (2014). Comparative 2D-DIGE analysis of salinity responsive microsomal proteins from leaves of salt-sensitive Arabidopsis thaliana and salt-tolerant Thellungiella salsuginea. J Proteomics. 2014 Jun 2. pii: S1874-3919(14)00288-7. doi: 10.1016/j.jprot.2014.05.018.
Hsueh et al. (2014). The chloroplast outer envelope protein P39 in Arabidopsis thaliana belongs to the Omp85 protein family. Proteins. 2014 Nov 17. doi: 10.1002/prot.24725. - Protocols
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Agrisera Western Blot protocol and video tutorials
Protocols to work with plant and algal protein extracts
Oxygenic photosynthesis poster by prof. Govindjee and Dr. Shevela
Z-scheme of photosynthetic electron transport by prof. Govindjee and Dr. Björn and Dr. Shevela - Reviews:
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Zeeshan Banday | 2019-08-29AS08351 worked well for me. 15ug Chloroplast and total extracts from leaves of Nicotiana benthamiana were incubated with 1:5000 dilution of anti-TOC75 at 4 degC O/N. The blots were then incubated with HRP- conjugated Ab @ room temperature for 2h. The signal was detected by chemiluminescence.Jun-Shian Chang | 2013-05-24The antibody can detect Toc75 easily in 30 ug pea-chlorplast protein. The antibody was 1000 times diluted and the western blot was visualized by alklaine phosphatase and NBT/BCIP method.
Accessories
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