In order to evaluate the immune reactivity of the constructed recombinant protein, 30 female BALB/c mice aged 6–8 weeks (10 animals per each group) obtained from Razi Vaccine and Serum Research Institute, Shiraz, Iran were immunized as follows. Group 1, were injected with PBS for 5 weeks as negative control, group 2, were administered with 200 μl inactivated agalactiae vaccine (contained bacterial suspension 2 × 109 CFU/ml, Horse serum, Saponin, Formaldehyde) (Razi Vaccine and Serum Research Institute, Shiraz, Iran) for two times at 2-week intervals and group 3, immunized with 60 μg recombinant fusion protein GST-PDHB-P80 five times at 2-week intervals. In groups 1 and 3, GST-PDHB-P80 and PBS were emulsified with the same volume of complete Freund’s adjuvant in the first immunization and with incomplete Freund’s adjuvant (Razi Vaccine and Serum Research Institute, Iran) in the following immunizations in a total volume of 200 μl per mouse. After bleeding, we used 1:100 diluted sera raised from the treated animals as primary antibody in Western blot analysis on the blotted GST-PDHB-P80. Goat Anti-Mouse IgG (H L)-HRP antibody (Bio-Rad, U.S.A) was applied at a ratio of 1 to 1000 as secondary antibody. The incubation with sera and secondary antibody performed at room temperature for two hours.
Goat anti mouse igg h l hrp antibody
Manufactured by Bio-Rad
Sourced in United States
Goat Anti-Mouse IgG (H+L)-HRP antibody is a secondary antibody conjugated with horseradish peroxidase (HRP). It is designed to detect and bind to mouse immunoglobulin G (IgG) antibodies, including both heavy and light chains.
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2 protocols using goat anti mouse igg h l hrp antibody
1
Evaluation of Recombinant Protein Immunity
2
Quantitative Analysis of FGF7 and FLT3L Proteins
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Equal amounts of concentrated CM (20 µl for 2×105 cells) from BM-MSCs, AT-MSCs and T-MSCs were loaded onto a 5% stacking/10% separating polyacrylamide gel, separated by electrophoresis, transferred to polyvinylidene difluoride (PVDF) membranes, blocked with 5% skim milk in TBST (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1% Tween-20) and incubated with primary antibodies overnight at 4°C. All primary antibodies were prepared by diluting in 3% BSA (Bovogen Biologicals) and 0.02% sodium azide (Sigma-Aldrich; Merck KGaA) in TBST. Anti-FGF7 mouse monoclonal antibody (sc-365440, 1:200, F-9, IgG1, κ), anti-FLT3L mouse monoclonal antibody (sc-365266, 1:200, F-6, IgG1, κ) and anti-β-actin mouse monoclonal antibody (sc-47778, 1:3,000, C4, IgG1, κ) were purchased from Santa Cruz Biotechnology, Inc. The PVDF membranes were washed 3 times for 10 min in TBST and incubated with goat anti-mouse IgG (H + L)-HRP antibody (#1706516, Bio-Rad Laboratories, Inc.) and diluted in TBST (1:3,000) for 1 h at room temperature. Following incubation, the membranes were washed 3 times for 10 min in TBST and developed using EZ-Western Lumi Femto (DoGenBio Co.). Images were obtained using ImageQuant LAS 500 (GE Healthcare Life Sciences). The pixel densities of the FGF7 and FLT3L bands were divided by the pixel densities of the corresponding β-actin bands for protein quantitation using UN-SCAN-IT-gel 6.1 software.
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