Cnbr activated agarose

The cysteine substituted Asn226Cys/Arg240Cys hCALHM2 and Arg52Cys/Tyr182Cys hCALHM2, as well as wild-type hCALHM2, were all C-terminally 1D4 tagged and expressed in the BV/Sf9 expression system under the CMV promoter. The membrane fraction were isolated and solubilized as above. The solubilized fraction was subjected to affinity purification by 1D4 antibody conjugated to CNBr-activated agarose (GE Healthcare). The resin was extensively washed and the protein eluted with wash buffer supplemented with 0.2 mg/ml 1D4 peptide. Samples were then either reduced with β-mercaptoethanol, left untreated, or treated with the oxidizing agent (1,10-phenanthroline) copper(II). After 30 min incubation on ice, 1 mM final concentration iodoacetamide was added to samples treated with (1,10-phenanthroline) copper(II). Samples were subjected to Western blot using anti-1D4 monoclonal antibodies (University of British Columbia) and anti-mouse Horseradish peroxidase-conjugated antibodies (GE Healthcare). Protein bands were detected by enhanced chemiluminescence on X-ray film (ECL kit; GE Healthcare). To verify that the in-membrane hCALHM2 assembly corresponded in size to detergent extracted hCALHM2, the membrane fractions containing Asn226Cys/Arg240Cys hCALHM2–1D4 were also oxidized with (1,10-phenanthroline) copper(II) prior to detergent solubilization.