The Transcreener®ADP Assay Kit contains all the components to detect ADP generated from any kinase or ATPase reaction. The Transcreener® ADP Assay Kit comes complete with ADP Antibody, ADP Far-Red Tracer, Stop and Detect Buffer and ADP. The user must supply kinase, substrates, and kinase reaction buffer.
Yes. The Transcreener® ADP Assay Kit is a universal assay and works well with both purified tyrosine and serine/threonine protein kinases in common kinase buffers. The utility of the assay is expanded to include non-protein kinases, such as those that use an alcohol, carboxyl, nitrogenous, or phosphate group as an acceptor. In fact, any reaction producing ADP is appropriate for this assay.
Because the assay is based on the generic detection of ADP produced in all kinase reactions, theoretically any type (or most concentrations) of kinase acceptor substrate can be used including peptides, proteins, nucleosides, sugars, and lipids. Phosphorylated proteins and peptides and inactive native proteins involved in cascading pathways (i.e. MAPK signaling pathways) may also work well in the Transcreener® ADP Assay.
Yes. The Transcreener® ADP Assay was developed to follow the progress of kinase and ATPase reactions. Kinases catalyze the transfer of a phophate group from ATP to a protein, peptide, lipid or small molecule substrate generating ADP, the invariant product. During ATPase reactions, the transfer of a phosphate group from ATP to a water molecule generates ADP, and thereby competes with the ADP tracer for binding to the monoclonal anti-ADP antibody.
The Transcreener® ADP Assay requires an instrument capable of performing fluorescence polarization (FP) and with sufficient sensitivity to work with 2.0 nM fluorescently labeled compound. Click here for Instrument Compatibility Table.
Corning® Microplates, catalog# 4514 (384 well, black, round bottom, low volume, polystyrene, non-binding surface) are suitable for the Transcreener® FP Assays.
For TR-FRET assays it is important to use assay plates that are entirely white with a nonbinding surface. We recommend Corning 384-well plate (catalog #4513).
FI assay plates should be entirely black. Corning plates catalog# 4514 are recommended for 384-well versions. For 96 well assay formats, Greiner half area plates catalog# 675076 should be used.
Bellbrook Labs has not exhaustively tested all available plates. However, plates that contain non- or low- binding surfaces are recommended as much to increase the efficiency of the reaction as for the detection of product produced during the enzyme reaction. Also, adding 0.01% Brij to the reaction buffer will further minimize proteins sticking to the plate.
An ADP/ATP Standard Curve enables the end user to convert experimental mP , FI, or TR-FRET values to µM ADP, which can then be used to calculate % ATP converted. The ADP/ATP Standard Curve mimics a kinase reaction by keeping the adenosine concentration constant (as ADP is produced, ATP is depleted accordingly). Instructions on how to prepare a Standard Curve are found in the Transcreener® ADP Assay Technical Manual.
Under the reaction conditions used in the Transcreener® ADP Assay, the ADP antibody is >100 fold selective for ADP over ATP. This selectivity is quite sufficient for a robust assay. However, to achieve maximum sensitivity and assay window, the [ADP Antibody] must be optimized for each starting [ATP].For greater detail on this topic please refer to a recent poster on kinase profiling.
This platform can be used for starting concentrations from 1 to 500 µM ATP (highest concentration tested). We offer Transcreener® ADP kit optimized for 1-100 µM ATP. For starting [ATP] > 100 µM call BellBrook Labs.
The Transcreener® ADP assay (as described in the protocol) has proven to be tolerant to many additives and buffers. Click here for a current list of compatible buffer conditions and tolerance data for phosphatase inhibitors, metal chelators, solvents, detergents, reductants and salts.
For a wide range of starting ATP concentrations, a conservative approach suggests a minimum of 10% conversion is required for a Z’ > 0.5.
Direct inhibition of the tracer/Ab complex by test compounds is theoretically possible. However, a far red tracer is utilized to reduce compound interference. Less than 0.3% of Gen-Plus Library interfered with ADP detection using the Transcreener® ADP Assay. Indeed, to date all data generated using known kinase inhibitors suggest a very specific interaction between the ADP far red tracer and the ADP antibody. See recent poster for selectivity information.
All homogenous fluorescent readouts have some probability of artifacts from compound fluorescence or light scattering due to insoluble compounds. These artifacts are reduced significantly by using fluorophores that are far red-shifted. The Transcreener® ADP Assay uses an AlexaFluor™ 633 fluor which is compatible with current instrumentation.
Wouldn’t this contribute to background signal? Certain kinases catalyze some level of non-productive ATP hydrolysis to the extent that water is able to get into the active site. However, the rates are generally low even in the absence of acceptor substrate and are even further reduced when acceptor substrate is present. If you are using it to screen for potential acceptor substrates, then background from ATP hydrolysis has to be taken into account on a case-by-case basis. We recommend a “no substrate” control (described in the technical manual) to detect non-productive ATP hydrolysis.
The Transcreener® ADP Assay measures the production of ADP from whatever source. Therefore, any impurity that causes ADP production such as a contaminating kinase, phosphatase or ATPase will interfere with accurate measurement of the desired kinase activity. Care should be taken to minimize these potential contaminants in both kinase and protein substrate preparations.