am performing an ELISA method to determine EC50 binding of several glycan binding proteins against IgG (it is N-glycosylated at N297 in the Fc region). I am having difficulties in the ff
finding the upper and lower limit. Most of the initial concentration ranges I have done, when plotted serial dilution of the glycan binding protein concentration i.e. 10ug/ml, 5ug/ml, 2.5ug/ml etc. I do not reach the upper or lower limit values
For some of the glycan binding proteins, I will get the lower limit but I haven't gotten it for any when it comes to the upper limit. The next best thing I did was to increase the concentration range, i.e., 20ug/ml, 10ug/ml, 5ug/ml etc. instead of 10ug/ml, 5ug/ml, 2.5ug/ml etc. However, since all the glycan binding proteins should have equal incubation time when I do colorimetric development with TMB/HRP, the higher concentrations start to precipitate at the desired incubation time.
How do I fix this? Do I try:
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