Transcreener HTS Assays
Universal, high throughput screening platform for enzymes based on direct detection of nucleotide enzyme products with far red FI, FP and TR-FRET readouts.
Transcreener assays enable you to screen more targets, both well characterized and emerging, faster and more efficiently.
- Four assays cover thousands of target enzymes, including any kinase, ATPase, or GTPase.
- Direct detection means less chance for compound interference.
- Far Red FP, FI and TR-FRET readouts validated on major multimode readers.
- Single step, mix and read format with 8 hour deck and signal stability for easy automation.
Thousands of cellular reactions use nucleotides, either alone or in combination with other substrates. Enzymes in the purinome alone – those that use adenine and guanine nucleotides – comprise 13% of the coding capacity of the human genome (ref). Of these, protein kinases have become highly validated drug targets, but lipid kinases, chaperone ATPases and GTPases as well as less obvious ATP-dependent enzymes such as Acetyl Co-A carboxylase are increasingly being targeted for therapeutic intervention.
Four assays cover thousands of target enzymes, including any kinase, ATPase, or GTPase.
Four Transcreener assays enable detection of thousands of different nucleotide dependent enzymes.
Transcreener is a universal assay method that can be used across entire families of nucleotide-dependent enzymes. Rather than using separate assays for a multitude of specific reaction products, such as phosphorylated proteins or lipids, a single nucleotide detection assay can be used for all of the enzymes that generate a common nucleotide product.
Direct detection means less chance for compound interference.
Transcreener Assay principle shown using the GDP FI Assay as an example. Displacement of the tracer from antibody by GDP relieves quenching resulting in increased fluorescence.
Direct detection of nucleotides is unique to the Transcreener platform. The method is based on the interaction of two detection reagents: an antibody and tracer. Displacement of tracer by the nucleotide being detected causes a change in its fluorescence properties. In the case of the TR-FRET and FI formats, the antibody is covalently labeled, whereas for FP it is unmodified. Transcreener assays have fewer reagents and a less complex mechanism than any other nucleotide detection assay, all of which require coupling enzymes. Coupling enzymes are used to convert a nucleotide to a product that can be used by a reporter enzyme to generate a signal. Each coupling and reporter enzyme is a potential target for the compounds being screened, which increases the risk of false positives or of missing a hit.
PKA titration using 20µM ATP showing sensitive detection of very low levels of ADP formation.
The Transcreener platform relies on highly specific antibodies that are able to differentiate between nucleotides on the basis of a single phosphate or even a methyl group. Selectivities for the product nucleotides vs. the substrates (e.g., ADP vs. ATP) range from 150-fold to over 1000-fold. Differentiation between closely related nucleotides is central to the technology as it allows detection of nucleotide enzyme products in the presence of an excess of the substrate nucleotide (e.g, ADP detection in the presence of excess ATP), a requirement for measuring enzyme initial velocity. All of the Transcreener assays generate Z’ values of greater than 0.7 at substrate conversion levels of less than 10% - usually far lower – over the full range of initial substrate concentration.
Far Red FP, FI and TR-FRET readouts validated on major multimode readers.
BellBrook's partners in the Transcreener Instrument Validation Program.
In addition to giving you flexibility of three detection modes, we have collaborated with the major suppliers of multimode readers to optimize instrument hardware and software settings for maximal performance with each of the Transcreener Assays. Successful validation requires a Z' of greater than 0.7 for nucleotide detection under initial velocity conditions (≤10% conversion). Optimal filter sets and instrument settings are summarized in the Technical Manual for each Transcreener assay, and more detailed information is available in Application Notes. This ensures that whatever fluorescent detection mode or reader you prefer, you will get robust results with Transcreener assay reagents.
Single step, mix and read format with 24 hour deck and signal stability for easy automation.
Transcreener assays are homogenous, single addition methods
Direct detection also means that the protocol is the simplest available: run your enzyme reaction, add Transcreener reagents with stop mix, and read plates. Some coupled methods require extra liquid addition and incubation steps, which complicates assay automation.
The Transcreener antibodies and tracers are very stable molecules, which allows the detection mix to be pre-mixed and stored for long periods at room temperature. Also, unlike the transient signals from some coupled enzyme assays, the Transcreener fluorescent signals persist at least over night – some for days - so plates can be read long after the detection reagents are added. These properties make Transcreener very easy to use in an automated HTS environment.