Anti gst tag mouse monoclonal antibody

Indirect immunofluorescence assay (IFA) was carried out to determine the cellular localization of BoSBP4. In brief, B. orientalis-infected and normal water buffalo erythrocyte smears were fixed in cold 50% acetone: 50% methanol (v:v) solution for 30 min at -20 °C and then incubated with the anti-rBoSBP4 mouse serum (1:200), the naïve mouse serum (1:200, negative control) or anti-GST tag mouse monoclonal antibody (1:1000, negative control, Proteintech Group) for 1 h at 37 °C. After washing three times with the cold PBS, smears were incubated with the second antibody of Alexa-Fluor® 594-conjugated goat anti-mouse IgG (1:1000 dilution; Invitrogen) for 1 h at 37 °C. For nuclear staining, smears were incubated with Hoechst (1:1000 dilution, Invitrogen). Another negative control consisted of the second antibody only without incubation with the primary antibody. Imaging was performed using fluorescence microscopy.

To evaluate if B. orientalis-infected water buffalo can generate antibodies against SBP4, rBoSBP4 was verified by SDS-PAGE and subsequently blotted on a polyvinylidene fluoride membrane (PVDF, Merck Millipore, Kenny, NJ, USA). The membrane was blocked with 0.05% Tween-20 in tris buffered saline (TBST) plus 5% skimmed milk and then probed separately with the serum from B. orientalis infected water buffalo (1:400) or normal water buffalo. A secondary antibody (1:2000) of horseradish peroxidase (HRP)-conjugated anti-bovine IgG (Bioss, Beijing, China) was used to identify the bound proteins on the blots.
To identify the native form of SBP4 in B. orientalis merozoite, lysates of B. orientalis-infected water buffalo erythrocytes were incubated with the anti-rBoSBP4 mouse serum. Briefly, lysates of B. orientalis-infected and uninfected water buffalo erythrocytes were separated on 12% SDS–PAGE gel, blotted onto a PVDF (Merck Millipore, Kenny, NJ, USA) and probed with the anti-rBoSBP4 mouse serum (1:400) or the serum of naïve mouse or anti-GST tag mouse monoclonal antibody (1:1000, Proteintech Group, Chicago, USA) as controls. The membranes were washed with TBST and then incubated with goat anti-mouse IgG/HRP (1:2000) as secondary antibodies (Bioss). Protein bands on membranes were visualized using the DAB method (ZSGB-BIO, Beijing, China).