I want to study the function of my interested kinase, and now I want to use dot mutant skill to block the kinase site. Since I do not have this gene knockout cell model, so if I transfect the dot mutant plasmids into a cell line with endegenouos gene expression, does the mutant plasmids will affect the endengous gene's function?
Dear Jun zang,
Obviously, If the expression of your exogenous gene is higher than endogenous it relatively affect towards inhibition of kinase activity.
Yes it affects because concentration of mutant plamids play an important role in the kinase activity
Hi there,
If there is no effect on endogenous gene expression due to the presence of transfected plasmid then kinase activity level should be only diluted by the presence of the inactive mutant form of the kinase. If activity is measured directly on a crude cell lysate, dilution effect will be only noticable if activity is actually expressed toward the amount of protein present in the assay (provided the mutant is abundant enough).
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