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Frequently Asked Questions

Below, you will find the answer to the most frequently asked questions about our Transcreener® and AptaFluor® products. If you still can't find the answer to your question, please contact us!

Interested in ordering an assay kit? Check out our Ordering Information page for pricing and shipping details.

FAQ's: General

Transcreener is a mix-and-read enzyme assay method that directly detects nucleotides produced by enzymes (e.g., ADP for kinases) and produces a far-red fluorescent signal. It is the simplest and most sensitive HTS-compatible method available for detecting nucleotide enzyme products. The platform relies on antibodies that selectively recognize enzyme products vs. substrates (e.g., ADP vs. ATP); direct detection of nucleotides without intervening steps decreases the potential for interference. The ability to detect an entire families of enzymes (e.g., kinases, ATPases GTPases, glycosyltransferases, phosphodiesterases, ligases, exonucleases) using a single assay method streamlines assay development and specificity profiling.  The outstanding stability of Transcreener reagents and assay signal make it a flexible and reliable platform for an automated screening environment.

No, the assays are designed for use with purified target proteins. Cell lysates have mixtures of nucleotides and nucleotide metabolizing enzymes that will cause interference with the assays.

The Transcreener Assays require an instrument capable of performing a fluorescence polarization (FP), fluorescence intensity (FI), or TR-FRET readout. AptaFluor Assays require an instrument capable of performing a TR-FRET readout.

Click here for Instrument Compatibility Table and Application Notes describing filters and settings for instruments that have been validated for Transcreener and AptaFluor assays. Please contact the manufacturer for more information regarding optimal instrument settings.

The three detection modes yield very similar results (e.g., The Transcreener® ADP2 Assay: A Universal Kinase Assay for Both HTS and Lead Discovery). We find that the FI and FP assay generally provide slightly better sensitivity and less variability than TR-FRET as well as better signal stability over long periods of time (> 8 hr).  However, choice of detection mode depends mainly on instrument capabilities and personal preferences.

Transcreener Assays can be used with 96-, 384- or 1536-well plates.  Though we have not exhaustively tested all available plates, the indicated Corning plates, which are available from BellBrook Labs, provide the most consistent assay performance. Other plates with non-binding surfaces can be used, but we have found that results are more variable than with the recommended Corning plates. Also, use of 0.01% Brij in buffers will further minimize non-specific binding of reagents to plates.

Type of PlateReadout ModeManufacturer & Catalog #
Half Area, Non-binding Surface, Black Polystyrene, Flat-bottom Assay PlatesFP & FICorning #4514 (384-well)
Corning #3686 (96-well)
Corning #3728 (1536-well)
Half Area, Non-binding Surface, White Polystyrene, Flat-bottom Assay PlatesTR-FRETCorning #4513 (384-well)
Corning #3642 (96-well)
Corning #3729 (1536-well)

Learn more about which assay plates to use in this article: Ensuring Assay Success: The Right Assay Plate Makes All The Difference

First, check to make sure that you are doing the following:

  • Allow the High and Low controls to equilibrate for at least 60 minutes at room temperature.
  • Check that you are using the correct antibody and tracer concentrations as indicated in the Technical Manual.
  • Ensure that your instrument is capable of measuring in the desired mode and that it is correctly set up (Transcreener and AptaFluor Assays: Instrument Compatibility).
  • Ensure that the proper plate is being used for the detection mode you are using.
  • Use nuclease free water for the preparation of the controls.

If you are having issues after following these steps, please contact us and one of our scientists will reach out with further assistance.

The dynamic range of the assays can be tuned for your specific application by adjusting the concentration of the Transcreener antibody or tracer.  For Transcreener FP and FI detection modes, the tracer concentration always remains constant, and the dynamic range of the assay is determined by the antibody concentration: lowering the antibody makes the assay more sensitive, increasing makes it less sensitive. For TR-FRET, the concentration of the Antibody-Terbium conjugate is invariant, and the tracer concentration sets the dynamic range in a similar fashion.  For each Transcreener assay, we provide an equation or table in the Technical Manual for determining the optimal antibody or tracer concentration, based on the concentration of substrate being used (e.g., the concentration of ATP in a kinase reaction) and assuming initial velocity reaction conditions (≤ 10% substrate consumption). However, if you are using non-standard reaction conditions or buffer components that may affect the antibody-tracer interaction, you should perform an antibody (FP, FI) or tracer (TR-FRET) titration to determine the optimal concentration, typically the EC70-EC80 concentration, for your specific application.

Our assays detect specific nucleotide enzyme products (e.g., ADP for kinases) with outstanding selectivity vs. substrate nucleotides (e.g., ATP).   Any reagents that change the concentration of the target analyte, whether directly or indirectly, can interfere with the assay.  For example, impure enzymes may contain contaminating nucleotidases or phosphatases that degrade the target nucleotide, resulting in decreased signal. A more common source of interference is contaminating nucleotides in substrate nucleotide preparations (e.g., contaminating ADP in ATP preps) which will cause high background signals. For these reasons, we provide high purity substrate nucleotides for Transcreener assays that undergo rigorous quality control testing. Care should be taken to minimize these potential contaminants in both enzymes and substrate preparations from other vendors. We also recommend utilizing Ultrapure Nuclease Free Water, such as Invitrogen Part #AM9930, to reduce the possibility of contamination by enzymes that are able to degrade nucleotides.

Most transferase enzymes catalyze some level of non-productive nucleotide hydrolysis to the extent that water can 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 the assay to screen for potential acceptor substrates, then background from nucleotide hydrolysis must be considered on a case-by-case basis. We recommend a “no substrate” control to detect non-productive nucleotide hydrolysis.

Use of far-red tracers (emission ≥ 650 nm) minimizes the interference from fluorescent compounds and light scattering. In addition, FP and TR-FRET are ratiometric readouts, which further reduces interference.

The following are the excitation and emission pairs for each of the fluorophores utilized in our assays:

Fluorophore
Excitation (nm)
Emission (nm)
AlexaFluor 633
631
650
AlexaFluor 594
590
618
HiLyte Fluor 647
650
673
ATTO 647
643
665
ATTO 633
629
657
DyLight 650
652
672

Though rare, compound interference may still occur in some cases, hence it is important to perform an interference screen to ensure that accurate data is being generated.  Compounds can be screened for interference in mock reactions lacking enzyme that mimic a completed enzyme reaction, e.g., 90% ATP and 10% ADP for a kinase reaction.

FAQ's: Transcreener ADP2 Assay

The Transcreener ADP2 assay is appropriate for any reaction producing ADP, namely enzymes in families such as Kinases, ATPases, and Helicases.

Below is a partial list of enzymes that we have validated for detection with the Transcreener ADP2 Assay. This is by no means meant to be a comprehensive list, and our customers have used the assay for many more ADP-producing enzymes, some of which can be found in publications.  

The Transcreener ADP2 antibody exhibits 160-fold selectivity for ADP vs. ATP, which enables sensitive detection of ADP in an excess of ATP. It can accommodate ATP concentrations from 100 nM to 1000 µM with the lower limits of detection listed below:

ADP Assay LLD Graphs in FP Readout
ADP Assay LLD Graphs in FI Readout
ADP Assay LLD Graphs in TR-FRET Readout
Sensitivity of the ADP Assay

To ensure that this selectivity is observed, we supply highly purified ATP with all Transcreener ADP2 assay kits and associated products. The antibody also recognizes other nucleotides diphosphates with similar affinity to that of ADP but has high selectivity vs. AMP and other related nucleotides.

For more information, please see the following poster: The Transcreener® ADP2 Assay: A Universal Kinase Assay for HTS and Lead Optimization

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, lipids, and so on. Phosphorylated proteins and peptides and inactive native proteins involved in cascading pathways may also work well in the Transcreener ADP2 Assay.

Reagents in the Transcreener ADP2 Assay Kits have been shown to have signal and deck stability of at least 24 hours at room temperature. We recommend aliquoting the reagents and storing them at their ideal temperature for best performance.

FAQ's: Transcreener AMP2/GMP2 Phosphodiesterase Assay

The Transcreener AMP2/GMP2 assay is appropriate for any reaction producing AMP or GMP, namely enzymes in families such as Phosphodiasterases, Ligases, and Synthetases.

Below is a partial list of enzymes that we have validated for detection with the Transcreener AMP2/GMP2 assay. This is by no means meant to be a comprehensive list, and our customers have used the assay for many more AMP or GMP-producing enzymes, some of which can be found in publications.

The Transcreener AMP2/GMP2 antibody exhibits outstanding selectivity for AMP and GMP vs related nucleotides. It has a lower limit of detection of 1 nM and the ability to accommodate substrate concentrations from 100 nM to 1000 µM.

Specificity of the AMP GMP Antibody Graph
Specificity of the AMP GMP Antibody Table
Sensitivity of the AMP GMP Assay Graph

FP Assay

Sensitivity of the AMP GMP Assay Graph in FP Readout
Sensitivity of the AMP GMP Assay Table in FP Readout

TR-FRET Assay

Sensitivity of the AMP GMP Assay Graph in TR-FRET Readout
Sensitivity of the AMP GMP Assay Table in TR-FRET Readout

Though not included in the kit, highly purified ATP, cAMP and cGMP are available for purchase to ensure that this selectivity is observed.  For more information please see the following posters:

Targeting ENPP1 for Cancer Immunotherapy: Development of an HTS

Development of a High Throughput Transcreener® Assay to Explore the Ectonucleotidase Enzyme Family

Because the assay is based on the generic detection of AMP and GMP produced in enzyme reactions, theoretically any type (or most concentrations) of acceptor substrate can be used including peptides, proteins, nucleosides, sugars, lipids, and so on.

Reagents in the Transcreener AMP2/GMP2 Assay Kits have been shown to have signal and deck stability of at least 24 hours at room temperature. We recommend aliquoting the reagents and storing them at their ideal temperature for best performance.

FAQ's: Transcreener GDP GTPase Assay

The Transcreener GDP assay is appropriate for any reaction producing GDP, namely enzymes in families such as GTPases, Gα proteins, Ras-like G proteins, GTPase Activating Proteins (GAPs), and guanine nucleotide exchange factors (GEFs). It is also able to detect the activity of Glyfcosyltransferases, such as fucosyltransferases and mannosyltransferases.

Below is a partial list of enzymes that we have validated for detection with the Transcreener GDP assay.  This is by no means meant to be a comprehensive list, and our customers have used the assay for many more GDP-producing enzymes, some of which can be found in publications

The Transcreener GDP antibody exhibits greater than 100-fold selectivity for GDP vs. GTP, which enables sensitive detection of GDP in an excess of GTP. It has a lower limit of detection of 20 nM and with the ability to accommodate GDP concentrations from 1 µM to 1000 µM.

GDP Assay Selectivity Graph
Dynamic Range GDP Assay

To ensure that this selectivity is observed, we supply highly purified GTP with all Transcreener GDP assay kits and associated products. For more information please see the following poster:

Interrogation of Modulators of GPCR and small GTPase Activity: Screening GAPs and GEFs with the Transcreener.

Because the assay is based on the generic detection of GDP produced in enzyme reactions, theoretically any type (or most concentrations) of acceptor substrates can be used including peptides, proteins, nucleosides, sugars, lipids, and so on.

Reagents in the Transcreener GDP Assay Kits have been shown to have signal and deck stability of at least 24 hours at room temperature. We recommend aliquoting the reagents and storing them at their ideal temperature for best performance.

FAQ's: Transcreener UDP2 Glycosyltransferase Assay

The Transcreener UDP2 Glycosyltransferase Assay is appropriate for any reaction producing UDP, namely enzymes in families such as Glucosyltransferases, Galactosyltransferases, N-acetylglucosyltransferases, N-acetylglucosaminyltransferases, Xylotransferases and Glucosylceramide synthases.

Below is a partial list of enzymes that we have validated for detection with the Transcreener UDP2 Assay. This is by no means meant to be a comprehensive list, and our customers have used the assay for many more UDP-producing enzymes, some of which can be found in publications.

The Transcreener UDP2 antibody exhibits exquisite selectivity for UDP vs. uridine or UDP sugar donors. It has a lower limit of detection of 150 nM and with the ability to accommodate UDP concentrations from 1 µM to 1000 µM.

Competition Curve with Various UDP-Sugars
Table with Various UDP-Sugars
UDP Standard Curves
UDP Robustness Table

Because the assay is based on the generic detection of UDP produced in enzyme reactions, theoretically any type (or most concentrations) of UDP donors can be used including UDP-glucose, UDP-galactose, UDP-GlcNAc, UDP-GalNAc, UDP-xylose, and UDP-glucuronic acid.

Reagents in the Transcreener UDP2 Glycosyltransferase Assay Kits have been shown to have the following signal and deck stabilities:

Assay
Signal Stability
Deck Stability
UDP FP
24 hours
24 hours
UDP FI
24 hours
8 hours

We recommend aliquoting the reagents and storing them at their ideal temperature for best performance.

FAQ's: Transcreener cGAMP cGAS Assay

The Transcreener cGAMP antibody exhibits outstanding selectivity with nanomolar sensitivity for cGAMP vs. ATP, GTP, cGAS Substrates and related molecules.

cGAS Assay Outstanding Selectivity Data

To ensure that this selectivity is observed, we supply highly purified ATP, GTP, and Interferon Stimulatory DNA with all Transcreener cGAMP cGAS assay kits. For more information please see the following posters:

Targeting cGAS for Type 1 Interferon-Driven Autoimmune Diseases

Targeting the cGAS-STING Pathway Using a Homogenous, HTS Compatible Transcreener cGAS Assay

Reagents in the Transcreener cGAMP cGAS Assay Kits have been shown to have the following signal and deck stabilities:

Assay
Signal Stability
Deck Stability
cGAMP cGAS FP
24 hours
24 hours
cGAMP cGAS TR-FRET
3 hours
2 hours

We recommend aliquoting the reagents and storing them at their ideal temperature for best performance.

FAQ's: Transcreener dAMP Exonuclease Assay

The Transcreener dAMP Exonuclease assay is appropriate for any reaction producing dAMP, namely enzymes in families such as Exonuclease.

Below is a partial list of enzymes that we have validated for detection with the Transcreener dAMP Assay. This is by no means meant to be a comprehensive list, and our customers have used the assay for many more dAMP-producing enzymes, some of which can be found in publications

Validated Targets
TREX1
WRN Exonuclease

The Transcreener dAMP antibody exhibits outstanding selectivity for dAMP vs. dCMP, dGMP and related molecules. It has a lower limit of detection of 1 nM and with the ability to accommodate dAMP concentrations from 10 nM to 100 µM.

Selectivity Curve dAMP Assay
Sensitivity Curve dAMP Assay

For more information please see the following poster: Interrogating TREX1 with the Transcreener dAMP Exonuclease Assay.

Because the assay is based on the generic detection of dAMP produced in exonuclease or other enzyme reactions, theoretically any type (or most concentrations) of DNA or other substrates can be used.

Reagents in the Transcreener dAMP Exonuclease Assay Kits have signal and deck stability of at least 24 hours at room temperature. We recommend aliquoting the reagents and storing them at their ideal temperature for best performance.

FAQ's: Transcreener EPIGEN SAH Methyltransferase Assay Kit

The Transcreener EPIGEN SAH Methyltransferase assay is appropriate for any reaction producing SAH. It utilizes the Transcreener AMP²/GMP² Assay with coupling enzymes that convert the SAH produced by enzymes into AMP for detection.

Below is a partial list of enzymes that we have validated for detection with the Transcreener EPIGEN SAH Methyltransferase Assay.  This is by no means meant to be a comprehensive list, and our customers have used the assay for many more SAH-producing enzymes, some of which can be found in publications.

Validated Targets
DNMT1
pCAF
Dot1L
PRMT1
EZH2
PRMT3
GLP
PRMT8
GCN5
SET8/4
G9a
SET7/9
MLL1
SMYD3
MLL4
SUV39H1
NSD2

The Transcreener EPIGEN SAH Methyltransferase Assay has a lower limit of detection of 30 nM and can accommodate SAM concentrations ranging from 100 nM to 50 µM.

EPIGEN Standard Curve
EPIGEN Z' Data

Because the assay is based on the generic detection of SAH produced in methyltransferase reactions, theoretically any type (or most concentrations) of substrate can be used. Please see the graph below for examples of AMP2 antibody concentration optimizations for use with different methyltransferase acceptor substrates.

EPIGEN Assay Optimization of Antibody

Reagents in the Transcreener EPIGEN Methyltransferase Assay Kit have been shown to be stable for more than 16 hours at room temperature after preparation. The assay also has a signal stability of up to 48 hours after addition of all reagents to the plate. We recommend aliquoting the reagents to ensure the best assay performance.

For more information, please see the following poster: Development and Validation of a Generic Fluorescence Methyltransferase Activity Assay Using the Transcreener AMP2/GMP2 Assay

FAQ's: AptaFluor SAH Methyltransferase Assay

The AptaFluor SAH Methyltransferase assay is appropriate for any reaction producing SAH, namely enzymes in the families such as Histone Methyltransferases and DNA/RNA Methyltransferases.

Below is a partial list of enzymes that we have validated for detection with the AptaFluor SAH Methyltransferase Assay.  This is by no means meant to be a comprehensive list, and our customers have used the assay for many more SAH-producing enzymes, some of which can be found in publications

The AptaFluor SAH riboswitch exhibits outstanding selectivity for SAH vs. related nucleotides. It has a lower limit of detection of 0.6 nM and with the ability to accommodate SAM concentrations from 100 nM to 5 µM.

AptaFluor Selectivity Curve
AptaFluor Z' and SAH Titration Data and Graph

To ensure that this selectivity is observed, we supply highly purified SAM with all AptaFluor SAH Methyltransferase assay kits. For more information, please see the following poster:

AptaFluor™ SAH: A Homogenous, Universal Methyltransferase Assay Based on a Microbial Riboswitch

Because the assay is based on the generic detection of SAH produced in methyltransferase reactions, theoretically any type (or most concentrations) of acceptor substrate can be used including peptides, histones, nucleosomes, DNAs, and RNAs.

AptaFluor Standard Curve with Various Substrates
AptaFluor RNA Titrations

Reagents in the AptaFluor SAH Methyltransferase Assay Kit are stable for at least 5 freeze/thaw cycles when stored under the recommended conditions. We have additionally seen that the SAH Detection Mix is stable for at least 5 freeze/thaw cycles after preparation. We recommend aliquoting the reagents to ensure the best assay performance.

For more information, please see the following poster:

AptaFluor SAH: A Homogenous Universal Assay for Histone, RNA, and DNA Methyltransferases. Case Study for PRMT5, MLL4, METTL3/14, and NSP14

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