Alkaline phosphatase conjugated anti rabbit igg antibody

To compare the capacity of FHR1*A and FHR1*B to compete with FH to bind C3b, FH (9 μg/ml) was immobilized on the surface of a microtiter plate (Thermo Fisher Scientific) at 4°C overnight. After blocking with 1% BSA/PBST at 37°C for 1 hour, serial dilutions of the recombinant FHR1*A or FHR1*B proteins (2.5 μg/ml-0.039 μg/ml) as well as C3b (0.25 μg/ml) were added to the plates. After shaking for 30 sec, the plates were incubated at 37°C for 1 hour. After washing with 0.1%PBST, FH-bound C3b was detected with a C3c polyclonal antibody (Dako), followed by incubation with an alkaline phosphatase-conjugated anti-rabbit IgG antibody as the secondary antibody (Sigma-Aldrich). The plates were then developed with alkaline phosphatase chromogenic substrate (Sigma-Aldrich), and the optical density was read at 405 nm.

Crude mitochondria were initially subjected to induction of PTP opening in 0.25 M sucrose or 0.125 M ionic osmoticum (NaCl, KCl, Choline-Cl), 10 mM MOPS-Tris (pH 7.4), 10 μM EGTA-Tris, 1 mM Pi-Tris and then the incubation mixture was collected from the cuvette and immediately used for the extraction of proteins at different timepoints. Samples were ultra-centrifuged at 100,000 g for 40 min by a Beckman L7-55 centrifuge (Ty 70 Ti rotor) and the mitochondria in the pellet were re-suspended in 50 mM Tris-HCl (pH 6.8). The soluble fractions in the supernatants were concentrated by 5000 MWCO VIVASPIN 6 (Sartorius) at 10,000 g (SM24 Sorvall rotor) for 40 min. Thirty micrograms of the concentrated proteins were later separated by 15% (w/v) SDS–PAGE and electroblotted onto a nitrocellulose membrane to detect the presence of the Cyt c. Blots were incubated with anti-Cyt c rabbit polyclonal antibody (1:1,000 dilution, from Agrisera). The reactive proteins were finally detected by nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate staining, after incubation with alkaline phosphatase-conjugated anti-rabbit IgG antibody (1:2,500 dilution, from Sigma).