Functional Assay
hIL-33 binding to hST2-Fc confirmed by pull down assay. a) pull down of h-IL-33 by hST2-Fc. b) pull down of hST2-Fc by h-IL-33-His. 5 ug of hST2-Fc (or control Fc protein), 2 ug of hIL-33-His (or control His protein), and protein G resin (or anti-His resin) were incubated in 0.5ml RIPA buffer overnight at 4 degrees C. The precipitates were separated by SDS-PAGE, electro-transferred onto NC membrane, and immunoblotted for the presence of hIL-33-His or hST2-Fc with anti-His HRP or anti-hIgG HRP, respectively.
Functional Assay
Specific interaction of human ST2 with recombinant human IL-33. An indirect competitive ELISA was performed as follows; 1) coat microtiter plate wells with hST2-Fc (10 ug/ml); 2) add a varying concentrations of hIL-33 with or without a hIL-33 mAb to the wells followed by washing; 3) add anti-FLAG HRP conjugated (1:2,000) to an enzyme; 4) After adding the TMB solution, incubate at RT in the dark for 10 to 45 minutes. Immediately read the plate at 450 nm.
Functional Assay
hIL-33 binding to hST2-Fc confirmed by pull down assay. a) pull down of h-IL-33 by hST2-Fc. b) pull down of hST2-Fc by h-IL-33-His. 5 ug of hST2-Fc (or control Fc protein), 2 ug of hIL-33-His (or control His protein), and protein G resin (or anti-His resin) were incubated in 0.5ml RIPA buffer overnight at 4 degrees C. The precipitates were separated by SDS-PAGE, electro-transferred onto NC membrane, and immunoblotted for the presence of hIL-33-His or hST2-Fc with anti-His HRP or anti-hIgG HRP, respectively.
Functional Assay
Specific interaction of human ST2 with recombinant human IL-33. An indirect competitive ELISA was performed as follows; 1) coat microtiter plate wells with hST2-Fc (10 ug/ml); 2) add a varying concentrations of hIL-33 with or without a hIL-33 mAb to the wells followed by washing; 3) add anti-FLAG HRP conjugated (1:2,000) to an enzyme; 4) After adding the TMB solution, incubate at RT in the dark for 10 to 45 minutes. Immediately read the plate at 450 nm.
Functional Assay
hIL-33 binding to hST2-Fc confirmed by pull down assay. a) pull down of h-IL-33 by hST2-Fc. b) pull down of hST2-Fc by h-IL-33-His. 5 ug of hST2-Fc (or control Fc protein), 2 ug of hIL-33-His (or control His protein), and protein G resin (or anti-His resin) were incubated in 0.5ml RIPA buffer overnight at 4 degrees C. The precipitates were separated by SDS-PAGE, electro-transferred onto NC membrane, and immunoblotted for the presence of hIL-33-His or hST2-Fc with anti-His HRP or anti-hIgG HRP, respectively.
Functional Assay
Specific interaction of human ST2 with recombinant human IL-33. An indirect competitive ELISA was performed as follows; 1) coat microtiter plate wells with hST2-Fc (10 ug/ml); 2) add a varying concentrations of hIL-33 with or without a hIL-33 mAb to the wells followed by washing; 3) add anti-FLAG HRP conjugated (1:2,000) to an enzyme; 4) After adding the TMB solution, incubate at RT in the dark for 10 to 45 minutes. Immediately read the plate at 450 nm.
Functional Assay
hIL-33 binding to hST2-Fc confirmed by pull down assay. a) pull down of h-IL-33 by hST2-Fc. b) pull down of hST2-Fc by h-IL-33-His. 5 ug of hST2-Fc (or control Fc protein), 2 ug of hIL-33-His (or control His protein), and protein G resin (or anti-His resin) were incubated in 0.5ml RIPA buffer overnight at 4 degrees C. The precipitates were separated by SDS-PAGE, electro-transferred onto NC membrane, and immunoblotted for the presence of hIL-33-His or hST2-Fc with anti-His HRP or anti-hIgG HRP, respectively.
Functional Assay
Specific interaction of human ST2 with recombinant human IL-33. An indirect competitive ELISA was performed as follows; 1) coat microtiter plate wells with hST2-Fc (10 ug/ml); 2) add a varying concentrations of hIL-33 with or without a hIL-33 mAb to the wells followed by washing; 3) add anti-FLAG HRP conjugated (1:2,000) to an enzyme; 4) After adding the TMB solution, incubate at RT in the dark for 10 to 45 minutes. Immediately read the plate at 450 nm.