Goat anti mouse conjugated to hrp

One-to-three μM recombinant proteins ( ~ 5 μg total protein) and 20 μg mosquito SGE were separated by SDS-PAGE for western blots. Proteins were transferred to a PVDF membrane (iBlot, Invitrogen, Waltham, MA, USA) that was blocked for ~ 2 h in blocking buffer (5% [w/v] instant non-fat dry milk (Carnation) in TBST). Membranes were incubated O/N at 4 °C with anti-glucosidase mouse serum (1:2000 in blocking buffer). The next day, membranes were washed with TBST (2 X for 10 min) and TBS (1 X for 10 min) and incubated at RT for 1-2 h with goat anti-mouse IgG conjugated to alkaline phosphatase (Sigma Aldrich, St. Louis, MO, USA; 1 mg/ml, diluted 1:10,000 in blocking buffer) or goat anti-mouse conjugated to HRP (Invitrogen, Waltham, MA, USA; 1:3000, diluted in blocking buffer). The membranes were again washed with TBST (2 X for 10 min) and TBS (1 X for 10 min). Blots were developed using Western Blue Stabilized alkaline phosphatase substrate (Promega, Madison, WI, USA) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, Waltham, MA, USA). All blots were imaged using an Azure 300 imaging system (Azure Biosystems, Dublin, CA, USA).