Representative Serine/Threonine Kinase Substrate Screening Kit Plate 1 using Akt1 Kinase. Akt1 was used to phosphorylate biotinylated substrate peptides. Vertical-axis is DELFIA® count. Rows are noted by letters A-H. Columns are noted by numbers 1-12. Values in columns 1, 4, 7, and 10 are DELFIA® counts for positive control biotinylated phosphopeptides (shown in yellow). Negative control values (nonphosphopeptides) for the array are found in columns 2, 5, 8, and 11 and shown in blue. Experimental values are found in columns 3, 6, 9, and 12 and shown in red. Positive hits on the array are noted along with partial peptide sequence that served as substrate.
Kinase of interest is incubated with biotinylated substrate peptides. Phosphorylated products are captured on streptavidin-coated plates. Cell Signaling Technology antibodies serve as primary detection reagents; DELFIA® Europium (Eu) anti-rabbit or anti-mouse chelates are used as secondary antibodies. Positive hits from Serine/Threonine Kinase Substrate Screening Kit (#7400) can be used in any subsequent peptide based kinase assay.