Enzyme Assays

Enzyme Assays Filtration-based enzyme assays using MultiScreenHTS 96- and 384-well filter plates Enzymes, such as those involved in signal transduction pathways, are critical to the control and regulation of virtually all cellular functions. It is not surprising, therefore, that signaling enzymes such as the protein kinases and phosphatases are under intense investigation in both basic research and drug discovery programs. Recent advances made in unraveling the mechanisms of these enzymes, in addition to the development of powerful high throughput screening technologies, have lead to more specific and reliable screening techniques. The MultiScreenHTS Assay System allows you to reliably perform these assays in one filter-bottomed, multiwell plate. This not only saves time, conserves samples, produces less solvent and radioactive waste, and enhances consistency, but it also makes complete automation easy for most popular plate-based assays. Because these techniques utilize a separations step (or steps), their use generally results in more specific, reliable "leads." Cell-based assays can especially lead to bioactivities more related to those found in vivo. Available in a wide variety of membrane and plate materials, the MultiScreenHTS system is extremely versatile. It can be used to perform numerous enzyme assays, such as kinases, phosphatases, proteases, and endonucleases, and a number of second messenger assays such as cAMP, cGMP, phosphodiesterases (PDE), Nitric Oxide (NO), Ca2+, and inositols. Different filter materials allow for low or high protein- and DNA-binding, ion-exchange, and solvent resistance. Recommended filter types for enzyme assays include: - Low protein-binding PVDF (HV, DV) - High protein-binding mixed cellulose esters (HA) - High protein-binding hydrophobic PVDF (IP) - Negatively charged phosphocellulose (PH) - Positively charged DEAE (DE) - Glass fiber (FB, FC) The assorted available filters, with inherently different retention and separation characteristics, can be exploited to implement fundamental separation techniques in both 96- and 384-well formats.

*NEW*
MultiScreen_HTS+ Hi Flow filter plates: improve washing efficiency and reduce data variability.


Protocol

1. Pre-wet and add protein kinase, 32P-ATP, substrate protein or peptide, and co-factors; incubate to allow PKC to transfer 32P from ATP to substrate protein or peptide 2. Add cold TCA for precipitation assay or phosphoric acid for cation exchange assay 3. Collect proteins/peptides on filter; wash; dry plate; and count

MultiScreen_HTS Filter Plates
The new HTS design for both 96- and 384-well filter plates features rigid sidewalls aligned for robotic gripper arms. They also provide ample surfaces for barcode labels. The plate design eliminates surface contact with individual well “drip directors.” A plastic skirt improves the vacuum seal when used with a Millipore MultiScreen^™_HTS vacuum manifold.

Use of 384-well plates offers the reagent saving benefits of assay miniaturization and less waste. Results with the 384-well plate are shown to be equivalent to 96-well results.