Example GST 6XHis tagged standard curve. GST-His tagged protein (provided with the kit) was diluted from 50 ng/mL to 0.7 ng/mL. Background was subtracted from data points prior to log transformation and curve fitting (log-agonist vs. response – variable slope). Sensitivity was calculated mathematically to be as low as 0.5 ng/mL.
Example GSK3B-His tagged standard curve. GSK3B-His tagged protein (not provided with the kit) was diluted from 5µg/mL to 0.07µg/mL and loaded on to the His plate. GSK3b was detected with a rabbit monoclonal antibody and labeled with the provided HRP label. Background was subtracted from data points prior to log transformation and curve fitting (log-agonist vs. response – variable slope).
Validation by Western blot. Capture antibody was tested by western blot against three His tagged proteins: GST recombinant protein loaded at 50ng/well (lane 1), GSK3b recombinant protein loaded at 50ng/well (lane 2) and Hexokinase 1 recombinant protein loaded at 250ng/well (lane 3). Blots were blocked with 5% Milk in TBST buffer, incubated overnight in anti 6XHis-tag primary antibody and labeled with GAM HRP.
This kit is effective with a wide variety of sample matrices.