MDH4 | Malate dehydrogenase 4 (cytoplasmic)

Immunoprecipitation is performed by using Dynabeads Protein A: briefly 100 µl suspension is washed with 200 µl TTBS (Tris Buffered saline, 50 mM Tris-HCl pH 7.6 and 165 mM NaCl with 0.1% Tween 80) using the magnetic stands for concentrating the magnetic beads. After wash the beads are preincubated with 20 µl primary antibodies in 180 µl TTBS at room temperature for 30 minutes (minimum 15 minues). A first wash is followed afterwards with 200 µl TTBS and hence a real incubation with 200 µl plant extract (supernatant 20,000 x g for 3 min.), 200µl of TTBS and further 50 µl YeastBuster reagent (Novagen) containing a mixture of detergents to break and solubilize the mitochondria membrane. This incubation at room temperature is allowed to be under mild shaking to allow the beads to be in suspension. Hence supernatant is aspirated away by the use of the magnetic stand and two further washesing steps with 200 µl TTBS are performed prior mixing with 100 µl SDS-Sample buffer.Antibody is also binding to recombinant MHD4 (see image below).

Malate dehydrogenase 4(cytoplasm)(EC:1.1.1.37) has catalytic activity for the conversion of (S) -malic acid + NAD+ = oxaloacetic acid + NADH.