Figure 7
Microtiter plate array peptides serve as substrates for p60c-src with specificity and concentration dependence Eight-well strips bearing six substrate peptide wells and two control wells were robotically synthesized ab initio, in microtiter plate format. All of the peptide substrates were initiated with Cys(Acm) to provide additional extension from the polylysine backbone and a common attachment site. The Cys-SH protecting group (Acm) remains in place and all peptides were acetylated at the amino terminus. Duplicate substrate strips were reacted with each 90-μL dilution of c-Src enzyme containing 3, 1, 0.3, and 0.1 Units of enzyme. The wells were reacted for 20' at 30°, washed with distilled water and assayed by antibody ELISA for the presence of phosphotyrosine using a mixture of antibodies described in Figure 5. Nonlinear regression plots were computed in Prism and error bars represent the means ± 1 SD, which ranged between 3% and 7% of mean values. Iterations for (ABL) did not converge. All data points were corrected by subtraction of values obtained from wells with no peptide.