Goat anti mouse igg

Possible FH binding protein(s) in the midgut membrane extract were detected by using the ligand blot analysis[11 (link)]. Briefly, membrane proteins were extracted by incubating the A. stephensi mosquito midguts in the membrane extraction buffer (containing the protein inhibitor cocktail) from the ProteoJET membrane protein extraction kit (Fermentas, K0321) for 2 h at 4°C with constant shaking. Extracted membrane proteins were recovered by centrifugation at 16,000g for 15 min at 4°C. A volume equivalent to 1 midgut per lane was then loaded onto 10% SDS-PAGE gel under non-reducing conditions. Resolved membrane proteins were transferred to nitrocellulose membranes and overlaid after a blocking step with 3% non-fat milk for 1h with either 20% HIS (a source of FH) or PBS (a negative control for FH binding) and incubated overnight at 4°C. After 5 washes in PBS-T, the membranes overlaid earlier with HIS were further incubated in either PBS (a negative control for binding of the secondary antibody) or with 2 μg/ml of the monoclonal anti-human FH antibody (131X). After a 1-hour incubation at RT and 5 washes in PBS-T, the membranes were incubated in a 1:10,000 dilution of goat anti-mouse IgG coupled to HRP (NFH822, Perkin Elmer life Sciences, Inc.). After a 1-hour incubation at RT and 5 washes in PBS-T, the blots were developed as described above.