Toc75 | Protein TOC75-3, chloroplastic, POTRA domain 3

AS08 351  |  Clonality: Polyclonal  |  Host: Rabbit  |  Reactivity: A.thaliana, N. tabacum, P. patens, P. sativum, S. oleracea, T. salsuginea, some  cross-Reactivity was observed for cyanobacteria including:  Synechocystis, Synechococcus and Thermosynechococcus sp.

Toc75 | Protein TOC75-3, chloroplastic, POTRA domain 3 in the group Plant/Algal Antibodies / Photosynthesis  / Chaperones/Transporters at Agrisera AB (Antibodies for research) (AS08 351)


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product information

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]


Pisum sativum TOC75; Predicted POTRA Domain #3; Expressed and purified in E. coli using the Impact System from NEB. Peptide confirmed by MALDI. Q43715

Host Rabbit
Clonality Polyclonal
Purity Serum
Format Lyophilized
Quantity 50 µl
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 Flow Cytometry (Flow cyt), Immunolocalization (IL), Western blot (WB)
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AS08 345 | anti- Toc75 | Protein TOC75-3, chloroplastic, POTRA domain 1 rabbit antibodies

Plant protein extraction buffer

Secondary antibodies

Additional information
application information
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)

Confirmed reactivity Arabidopsis thaliana, Nicotiana tabacum, Physcomitrella 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, Nicotiana tabacum, Noccaea caerulescens, Populus trichocarpa, Ricinus communis, Theobroma cacao, Zostera marina, Vigna radiata, Vitis vinifera
Not reactive in No confirmed exceptions from predicted reactivity are currently known.
Additional information Antibody detects Toc75 POTRA domain 3 as purified protein, in chloroplast fraction and in crude envelope fraction.
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
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.
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.

application example

Western blot using anti-Toc75 POTRA3 antibodies

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 in Promega ECL Western Blotting Substrate 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

western blot using anti-TOC75 POTRA3 domain antibodies

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 HRP substrate Peroxide solution & luminol detection reagent according to the manufacturers instructions (Millipore). Exposure time was 600 seconds.

Ashita Dave and Barry D. Bruce (UTK)

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