VDAC1 | Voltage-dependent anion-selective channel protein 1
AS07 212 | Clonality: Polyclonal | Host: Rabbit | Reactivity: A.thaliana, di and monocots | Cellular [compartment marker] of mitochondrial outer membrane
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29 kDa (for Arabidopsis thaliana)
Arabidopsis alpina, Aundo donax, Brachypodium distachyon, Brassica campestris, Brassica napus, Brassica rapa subsp. pekinensis, Capsella rubella, Citrus clementina, Eutrema salsugineum, Glycine max, Glycine soja, Gossypium arboreum, Hoedum vulgare var. distichum, Jatropha curcas, Medicago truncatula, Mesembryanthemum crystallinum, Morus notabilis, Nicotiana tabacum, Phaseolus coccineus, Phaseolus vulgaris, Pisum sativum, Plantago major, Prunus persica, Ricinus communis, Solanum lycopersicum, Solanum tuberosum, Sorghum bicolor, Theobroma cacao, Triticum aestivum, Vitis vinifera, Zea mays
Chlamydomonas reinhardtii, Glycine max, Zea mays, diatoms, Saccharomyces cerevisiae
Crude membrane proteins were separated on 12% SDS-PAGE and blotted 1h to PVDF. Blots were blocked immediately following transfer in 5% blocking reagent (BioRad, 170-6404) in 50 mM Tris, 150 mM sodium chloride pH 7.5 with 0.1% (v/v) Tween-20 (TBS-T) for 1h at room temperature with agitation. Blots were incubated in the primary antibody in 1: 5000 dilution for over-night at 4°C with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed 3 times for 5 min in TBS-T at room temperature with agitation. Blots were incubated in secondary antibody (Goat anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:5000 in 0.2% blocking solution for 1h at room temperature with agitation. The blots were washed as above and developed for 1~2 min with chemiluminescent detection reagent, according the manufacturers instructions. Images of the blots were obtained using a CCD imager (LAS4000 GE) and by ImageQuant software (GE).
Fixation and Immunolocalization
(A) full confocal stacks; (B) Single confocal section
Pollen tubes were fixed in 400 μM 3-maleimodobenzoic acid N-hydroxysuccinimide ester (MBS, Pierce) for 6 min at 20ºC, followed by 2% formaldehyde (1 h, 4°C). Cells were washed three times in 1x TBS then once in MES buffer (15 mM MES, pH 5.0), then incubated in 0.05% cellulose/0.05% macerozyme with 0.1% Triton X-100 in MES buffer containing 0.1 mM PMSF and 1 % BSA for 15 min. Cells were washed once in MES, then twice in TBS and then incubated in blocking solution (1% BSA in TBS) for 30 min at room temperature. Pollen was incubated with anti-VDAC1 antibodyies diluted in blocking solution (at 1:500) overnight at 4°C. Following TBS washes pollen was then incubated with the secondary antibody for 1.5 h at room temperature followed by further TBS washes. Pollen tubes were mounted on slides with 5 μL of Vectashield + DAPI (Vector Laboratories, USA) and coverslips sealed with nail varnish.
Method taken from Poulter et al (submitted) Actin-binding proteins implicated in formation of the punctate actin foci stimulated by the self-incompatibility response in Papaver . Submitted to Plant Physiology.
Courtesy Professor Noni Franklin-Tong, University of Birmingham, UK
Cellular [compartment marker] of mitochondrial outer membrane for western blot.
VDAC1 protein (called also Synonymes: At3g01280, outer mitochondrial membrane protein porin 1, T22N4_9, T22N4.9, VDAC 1, Voltage-dependent anion-selective channel protein 1, voltage-gated ion-selective channel) forms a channel through the cell membrane for diffusion of small hydrophilic molecules. Evolutionary origin of VDAC protein is not clear and their structure and properties are quite different making those proteins only conceptually like porins (Clausen et al. 2004).
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