Gamma CA | Gamma Carbonic anhydrases
AS17 4114 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana, Oryza sativa
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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).
Gamma CAH (Gamma Carbonic anhydrase) proteins are localized in the inner mitochondrial membrane. They are in a range of 25-30 kDa.
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