Proteins resolved on 10% SDS-PAGE were further transferred to nitrocellulose membrane using Mini Protein III equipment (Bio-Rad Laboratories, Richmond, CA, USA) for Western blot analysis. After blocking with phosphate-buffered saline with tween® 20 (PBST) containing 5% skimmed milk for 1 h at room temperature. Membrane was incubated with 1:2000 diluted anti-His tag antibody (AbD Serotec., Kidlington, UK) in 5% skimmed milk at 4 °C for overnight. After washes with PBS-T (PBS containing 0.1% tween 20) buffer, the filter was then incubated with 1:10 000 diluted secondary antibody, goat anti-mouse IgG conjugated HRP (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA), for 1 h at room temperature. After several times of PBS-T washes to remove the unbound antibodies, the signal was detected by TMB reagent (KPL, Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD, USA).
Anti his tag antibody
Manufactured by Bio-Rad
Sourced in United Kingdom
The Anti-His tag antibody is a laboratory reagent used for the detection and purification of proteins that have been engineered to contain a histidine (His) tag. The antibody specifically binds to the His tag, allowing for the identification and isolation of the tagged protein from complex mixtures.
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Lab products found in correlation
4 protocols using anti his tag antibody
1
Western Blot Analysis of His-tagged Proteins
2
Vero Cell-Based TcdA Binding Assay
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Vero cells seeded in T-75 flasks containing VP-SFM/4 mM glutamine were allowed to grow to 80% confluence at 37°C. An aliquot of resuspended cells (5 × 105 cells/mL) was mixed with either 80 μg/mL TcdA rRBD or its fragments at 37°C for 5 min. After washing with cold PBST, 1 μg/mL of either the anti-TcdA antibody PCG-4 or an anti-His tag antibody (AbD Serotec, Oxfordshire, UK) was added to the cells, and the mixture was incubated on ice for 30 min. After washing twice with cold PBST, 1 μg/mL FITC-conjugated secondary antibody (Sigma-Aldrich, St. Louis, MO) was added and mixed for surface staining. Before flow cytometry analysis, propidium iodide (Sigma-Aldrich) was added to assess cell viability.
3
CEA-Binding Capacity of Immunotoxins
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The ability of IMTXCEAαS, IMTXTRICEAαS and their scFv counterparts to bind human CEA was studied by ELISA as previously described27 (link). Plates (Nunc A/S. Roskilde, Denmark) were coated with CEA (0.25 µg/well), washed and blocked with 5% BSA in PBS. Then, 100 µl with the corresponding concentration of the different constructions were added and incubated for 1 hour at room temperature. After three washes, the wells were incubated for one more hour at room temperature with an anti His-tag antibody (BioRad). Washes were repeated and HRP-conjugated goat anti-mouse antibody was added for another hour at room temperature. Then, the plate was washed and developed with the corresponding substrate. Antigen-binding titration was performed with serial dilutions of the purified immunotoxins. Three independent replicates were conducted to calculate the average values.
4
Immunoblot Analysis of Malaria Antigens
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Briefly, total antigens (2 μg/lane) from uninfected bovine RBC or Mo7 iRBC (2% percentage of parasitized erythrocytes, PPE) were ran through 4-20% TGX™ gels (Bio-Rad Laboratories, Hercules, CA), using 5X Sample Buffer containing 10% 2-Mercaptoethanol (GenScript, Piscataway, NJ, USA). Antigens were then transferred to nitrocellulose membranes using iBlot 2™ (Invitrogen, Waltham, MA), and the membranes were blocked 1 hour in 5% milk. Membranes were then washed one time in 1xPBS with 0.1% Tween 20 (PBS-T) and incubated with a 1:500 dilution of either pre-immune rabbit sera or rabbit anti 15-mer antibody serum in 5% milk for one hour rocking at room temperature. After that, membranes were washed three times using PBS-T and incubated with a 1:1000 dilution of anti-rabbit IgG peroxidase labeled secondary antibodies (SeraCare, Milford, MA) in 5% milk for 30 minutes rocking at room T°. Membranes were then washed again three times in PBS-T and incubated with Prometheus Protein Biology Products 20-300B ProSignal® Pico ECL Reagent (company)?. Visualization of immune complexes was performed using the Azure™ Imaging System (Azure Biosystems, Dublin, CA). The immunoblot in Figure 1C
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