Cells from the spleen of a CCL-PR2/N (Figure 1 B) immunized mouse were fused with PAI mouse myeloma cells as a fusion partner. Hybrids were selected in hypoxanthine-aminopterin-thymidine (HAT) medium, and B-cell hybridoma lines secreting peptide-specific IgG were identified by peptide ELISA. For large-scale mAb production, cloned hybridoma cell lines were cultured in 175 cm2 flasks and mAbs were purified by protein A affinity chromatography (HiTrap rProtein A FF, Amersham Biosciences AB, Uppsala, Sweden). Purified mAbs were dialyzed against PBS, sterile-filtered, and stored at −80 °C.
Hitrap rprotein a ff
Manufactured by Cytiva
Sourced in Sweden, United States
HiTrap rProtein A FF is a chromatography column designed for the purification of antibodies and antibody fragments. It contains recombinant Protein A ligand immobilized on agarose beads, which allows for the selective capture and separation of immunoglobulins from complex samples.
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Lab products found in correlation
2 protocols using hitrap rprotein a ff
1
Monoclonal Antibody Production Protocol
2
Rabbit Antiserum Production for FSHR
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Antiserum against FSHR was raised in New Zealand white rabbits. Blood (1.5 mL) was collected prior to immunization to obtain basal serum. This basal serum was used as negative control. Each rabbit was then subcutaneously injected with a mixture of 1 mg of purified MBP-FSHR4 protein mixed with an equal volume of Freund's complete adjuvant (Sigma-Aldrich, St. Louis, MO, USA) on the back and proximal limbs (100 μL/site). Three booster injections were given with 0.5 mg recombinant protein each in incomplete Freund's adjuvant (Sigma-Aldrich) at 3-week intervals. Two weeks after the last injection, blood was collected and kept at 37 °C for 1 h and at 4 °C for 3-4 h to allow blood clotting. The antiserum was collected by centrifugation (3000×g for 10 min), purified using an affinity column (Hi Trap rProtein A FF, Amersham, USA) and stored at -20 °C until further use.
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