Quantitative measurement of proteins by western blotting with Cy5 (TM)-coupled secondary antibodies

The concentration of proteins in cells is an important parameter that deter mines how a protein will interact with other proteins or pharmacological ag ents. Recent developments in Western blotting techniques have now made this a method of choice to measure protein concentration in complex solutions s uch as total cell extracts. We show that detection of Cy5 TM-coupled second ary antibodies by PhosphorImager analysis produces signals that approach li nearity with respect to protein concentration over a 20-fold range. We used this technique to estimate cellular levels of zyxin, which is an important protein component of the actin cytoskeleton in mammalian cells. By produci ng specific protein standards based on sequences that are available from pu blic databases, it is now possible to estimate the concentration of almost any protein by this technique.