Horseradish Peroxidase Case Study
Materials and methods
Chemical and immunochemicals
Daidzin (DZ, ≥99%), daidzein (≥97%), and glycitein (≥95%) were purchased from Fujicco Co. (Kobe, Japan). Genistin (≥98%) and genistein (≥98%) were obtained from Wako Pure Chemicals Industries (Osaka, Japan), while puerarin (≥98%) and glycitin (≥99%) were obtained from Tokyo Chemical Industry Co. (Tokyo, Japan) and LC Laboratories (MA, USA), respectively. Ovalbumin (OVA, ≥98%) was purchased from Sigma-Aldrich (Steinheim, Germany). Horseradish peroxidase (HRP)-conjugated goat IgG to mouse IgG Fc and HRP-conjugated mouse IgG against T7-Tag were individually purchased from Organon Teknika Cappel Products (PA, USA) and Novagen (MA, USA), respectively. Toyopearl CM-650M cation exchange resin was …show more content…
The hemolymph containing DZ-scFvs were pooled and subjected to cation exchange chromatography. Cation exchanger TOYOPEARL CM-650M (20 mL; Tosoh Corp.) was packed and equilibrated with starting buffer [10% (v/v) glycerol in 50 mM Tris–HCl, pH 6.8]. The hemolymph treated with starting buffer and proteinase inhibitors was centrifuged to remove insoluble aggregates before the clear solution was subjected to cation exchanger. Unbound proteins were washed out using starting buffer, and then the bound proteins was eluted with starting buffer containing a gradient concentration of NaCl from 0 to 300 mM. Each fraction (20 mL) was collected and analyzed using iELISA and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to evaluate the presence of …show more content…
The same procedures that were used in the iELISA were used until the blocking step. In the competitive step, serial concentrations of DZ in 20% (v/v) methanol in water (50 µL) were added to each well, and then purified DZ-scFvs (diluted in T-PBS) was added (50 μL). An hour later, the plate was washed three times with T-PBS, and then bound DZ-scFvs were allowed to react with a diluted solution (1:5000) of HRP-conjugated mouse IgG against T7-Tag (100 µL/well) for 1 hour. In the last step, the substrate solution (100 µL/well) was added and incubated for 15 min. The developed color was then measured using a microplate reader with an absorbance at 405
Chemical and immunochemicals
Daidzin (DZ, ≥99%), daidzein (≥97%), and glycitein (≥95%) were purchased from Fujicco Co. (Kobe, Japan). Genistin (≥98%) and genistein (≥98%) were obtained from Wako Pure Chemicals Industries (Osaka, Japan), while puerarin (≥98%) and glycitin (≥99%) were obtained from Tokyo Chemical Industry Co. (Tokyo, Japan) and LC Laboratories (MA, USA), respectively. Ovalbumin (OVA, ≥98%) was purchased from Sigma-Aldrich (Steinheim, Germany). Horseradish peroxidase (HRP)-conjugated goat IgG to mouse IgG Fc and HRP-conjugated mouse IgG against T7-Tag were individually purchased from Organon Teknika Cappel Products (PA, USA) and Novagen (MA, USA), respectively. Toyopearl CM-650M cation exchange resin was …show more content…
The hemolymph containing DZ-scFvs were pooled and subjected to cation exchange chromatography. Cation exchanger TOYOPEARL CM-650M (20 mL; Tosoh Corp.) was packed and equilibrated with starting buffer [10% (v/v) glycerol in 50 mM Tris–HCl, pH 6.8]. The hemolymph treated with starting buffer and proteinase inhibitors was centrifuged to remove insoluble aggregates before the clear solution was subjected to cation exchanger. Unbound proteins were washed out using starting buffer, and then the bound proteins was eluted with starting buffer containing a gradient concentration of NaCl from 0 to 300 mM. Each fraction (20 mL) was collected and analyzed using iELISA and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to evaluate the presence of …show more content…
The same procedures that were used in the iELISA were used until the blocking step. In the competitive step, serial concentrations of DZ in 20% (v/v) methanol in water (50 µL) were added to each well, and then purified DZ-scFvs (diluted in T-PBS) was added (50 μL). An hour later, the plate was washed three times with T-PBS, and then bound DZ-scFvs were allowed to react with a diluted solution (1:5000) of HRP-conjugated mouse IgG against T7-Tag (100 µL/well) for 1 hour. In the last step, the substrate solution (100 µL/well) was added and incubated for 15 min. The developed color was then measured using a microplate reader with an absorbance at 405