Gamma CA | Gamma Carbonic anhydrases
AS17 4114 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana
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|Recommended dilution||1 : 1000 (WB)|
|Expected | apparent MW||9.97 KDa (gamma-CA1), 30KDa (gamma-CA2) strongest band, 27.83 (gamma-CA3), 25.08 KDa Arabidopsis thaliana proteins|
|Confirmed reactivity||Arabidopsis thaliana|
|Predicted reactivity||Arachis duranensis, Brachypodium distachyon, Brassica oleracea, Brassica rapa, Camelina sativa, Capsella rubella, Citrus sinensis, Daucus carota, Erythranthe guttatus, Fragaria vesca subsp. vesca, Gossypium hirsutum, Jatropha curcas, Juglans regia, Lupinus angustifolius, Malus x domestica, Medicago truncatula, Nelumbo nucifera, Oryza brachyantha, Populus euphratica, Prunus mume, Pyrus x bretschneideri, Raphanus sativus, Sesamum indicum, Setaria italica, Solanum tuberosum, Tarenaya hassleriana, Theobroma cacao, Triticum aestivum, Zea mays, Zostera muelleri
|Not reactive in|
|Selected references||Kühn et al. (2015). Complete Mitochondrial Complex I Deficiency Induces an Up-Regulation of Respiratory Fluxes That Is Abolished by Traces of Functional Complex I. Plant Physiol. 2015 Aug;168(4):1537-49. doi: 10.1104/pp.15.00589. Epub 2015 Jul 1.|
Mitochondrial proteins (1mg) from Arabidopsis thaliana ecotype Col-0 cell suspensions were separated by 2D BN/SDS-PAGE (Klodmann et al., 2011; Plant Physiology 157: 587-598). After gel electrophoresis, proteins were transferred to nitrocellulose membrane using semi-dry conditions (90 min; 0.8mA per cm2 of gel). After washing once in TBS-T buffer (10 min), blot was blocked overnight at 4ºC with 10% p/v milk in TBS-T. Blot was washed three times with 0.5% p/v milk in TBS-T (10 min each) and incubated 1 hour with primary antibody (1:1000 in TBS-T, 3% BSA and 0.02% NaN3). Washing was carried out three times with TBS-T, 0.5% BSA, 10 min each. Then blot was incubated 1 hour with secondary antibody (Anti-rabbit IgG conjugated to alkaline phosphatase at a dilution of 1:10 000) in TBS-T, 3% BSA, 0.02% NaN3). After washing (as above), blot was equilibrated 5 minutes in AP buffer (Tris-HCl 1M pH 9.5; 5M NaCl; 4M MgCl2). Finally, revealing was developed by incubation in AP buffer; 110 µg/ml NBT; 75 µg/ml BCiP. Reaction was stopped by discarding revealing solution and adding distilled water. Upper lane: 1D BN gel electrophoresis of mitochondrial proteins, with respective complexes.
Lower gel: second dimension, where mitochondrial proteins of complexes were separated. Blot section (indicated as dashed square in gel) reveals mainly two bands (around 30 kDa- arrows), corresponding to both gamma-CA2 forms. The smear along the 30 kDa line is interpreted as fragments of complex I containing CA2 protein (Perales et al., 2005; Journal of Molecular Biology, 350: 263-277).
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