SignaTECT DNA-Dependent Protein Kinase Assay System

Convenient, 1-Hour Kinase Activity Assays

The SignaTECT® Protein Kinase Assay Systems provide a rapid, radioactivity-based method for detection of kinase activity. The method is based on capture of phosphorylated labeled substrates on the SAM²® Biotin Capture Membrane, which offers low background and high signal-to-noise ratios, even for complex samples such as crude cell extracts, while retaining the high substrate capacity necessary to maintain optimum reaction kinetics.

Each SignaTECT® System contains a highly specific biotinylated peptide substrate for the appropriate kinase as well as the necessary reaction components. The researcher must supply [?-³²P]ATP.

Method Overview

After the kinase reaction is complete, the biotinylated, ³²P-labeled substrate is recovered from the reaction mix using the SAM²® Biotin Capture Membrane. After samples are spotted onto the SAM²® Membrane a series of short washes are performed to remove nonspecific label. The process is complete in less than 1 hour. The SAM²® Membrane is prenumbered and partially cut so that individual squares can be easily identified, separated and placed in scintillation vials. Alternatively, the intact SAM²® Membrane can be analyzed by phosphorimaging or conventional autoradiography.

Advantages of the SAM²® Membranes

• High binding capacity and high specificity characteristics produce lower backgrounds and higher signal-to-noise ratios compared to the traditional P81 phosphocellulose method of capture and measurement.
• The perforated and numbered membrane allows researchers to measure from 1 up to 96 kinase reactions and does not require as much "hands-on" manipulation as other methods used to measure kinase activity.
• Can be used under a variety of buffer/reaction conditions (e.g., cell extracts), which many other methods do not allow.
• High binding capacity allows use of the SignaTECT® Systems for kinetic studies.