Thermo Scientific™ LanthaScreen™ TR-FRET ERR gamma Coactivator Assay Kit, goat

Yes, a biotinylated substrate can be used in a LanthaScreen assay provided you have fluorescein-labeled streptavidin (available from Thermo Fisher Scientific Cat. No. SA100-02). A typical assay should contain 200-400 nM biotinylated substrate in the kinase reaction, and 1-2 nM Tb-labeled antibody and 50 nM fluorescein labeled streptavidin to detect the product. These conditions will need to be optimized for your particular system, but the recommended amounts are a good starting point. This format does, however, increase the cost associated with screening because of the additional component compared to the directly labeled substrate format. Also, it adds another layer of complexity with the additional component requiring optimization.

Try both or check our data to see if we have run your kinase of interest against these substrates (see reactivity chart at thermofisher.com/lanthascreen). In general, either substrate will work for a given tyrosine kinase. However, one of the substrates will probably show a lower EC50 with the kinase (i.e,. be a better substrate for the kinase), allowing less kinase to be used in the assay. Additionally, one substrate may show a larger assay window than the other (usually, but not always, Poly-GAT shows a larger window). However, either substrate will give an assay with an excellent Z' value.

If you want a specific peptide substrate, we offer two synthetic peptides determined to be effective substrates for src-family kinases; peptide 1 (YIYGSFK) and peptide 2 (KVEKIGEGTYGVVYK). Peptide 1 was a random peptide identified to be a pp60c-srcsubstrate and peptide 2 is a sequence from p34cdc2. Alternatively, fluorescein-labeled peptide substrates can be readily synthesized by us to suit your particular needs. In addition to peptide substrates, we offer fluorescein-labeled Poly-GT and Poly-GAT. We have had 100% success rate with these substrates to-date when assaying tyrosine kinases (TKs).

This depends on the volume of the assay and the concentration of antibody used. At 2 nM antibody concentration in a final volume of 20 µL, 25 µg of antibody will run >4000 assays, and 1 mg of antibody will run >165,000 assays.

In general, we recommend 2 nM as the final concentration of antibody in the detection reaction. The molecular weight of a Tb-labeled antibody is 150,000 Da so a concentration of a 2 nM solution corresponds to 0.3 µg/mL. This is a 1:3,300 dilution of a 1 mg/mL stock solution.

There are a couple of different ways to directly label your antibody. First, you can have our custom labeling service prepare the material for you. You send us 1 mg of material or more and we will perform the labeling reaction for you. As an alternative to that, you may buy our terbium chelate in an amine or thiol reactive form and perform the labeling reaction in your lab.

Yes, you may “indirectly” label your primary antibody by using a terbium- labeled, species-specific anti-IgG antibody. As a starting point for further optimization, we recommend a final concentration of 2 nM primary and 5 nM terbium-labeled secondary antibody in the assay well. The reaction conditions can be further optimized by increasing and/or decreasing the amount of terbium-lableled secondary antibody to determine the optimal amount to use. Check with the supplier of your primary antibody to determine its stock concentration. While in a majority of cases, the unlabeled primary and terbium-labeled secondary antibody may be pre-mixed prior to addition to the assay well, in some cases, separate additions (primary first, followed by secondary) are required. In general, assays using directly labeled antibodies may come to equilibrium faster or have a more stable signal (longer read window) than assays that use indirect labeling techniques. Also, although the response window (Z') is often acceptable when using an indirect labeling strategy, it may sometimes be improved by using a directly labeled primary antibody.

For tyrosine kinase assays, we offer four different terbium-labeled anti-phosphotyrosine antibodies. When using fluorescein-labeled poly-GAT or poly-GT, performance using the Tb-labeled PY20 antibody is excellent. When using specific peptide substrates, performance may vary between the different varieties of anti phosphotyrosine antibodies, and as such we recommend evaluating the assay with a selection of antibodies in order to find the antibody that gives optimal performance. However, for tyrosine kinase assays, any of the anti-phosphotryrosine antibodies will likely perform well.

For serine/threonine kinases, we provide a reactivity chart at thermofisher.com/lanthascreen which shows the performance of various kinases using suitable substrate/antibody pairs.

In general, 200 nM to 1 µM peptide or physiological protein substrate is often used in a LanthaScreen kinase assay. We recommend starting with 200 or 400 nM in the assay reaction. The molar concentration of substrate can be calculated from the molecular weight of the substrate, by dividing the mg/ml concentration by the molecular weight. This will give a molar concentration of the substrate. Multiplying by 1,000,000 will give a micromolar concentration. The molecular weight of each substrate can be found on the certificate of analysis for each individual substrate or on our website.

[Substrate] in µM = mg/mL/(molecular weight x 1,000,000)

The amount of ATP to use depends on the goals of the specific assay. In an assay designed to identify ATP-competitive inhibitors, the assay is typically run at the ATP Km apparent. To determine the ATP Km apparent, perform the assay using a serial dilution of ATP and a constant amount of kinase and substrate. The EC50 value of this curve is the ATP Km apparent. Now, using the determined concentration of ATP and the same amount of substrate, run the assay using a dilution series of kinase. From the kinase titration curve, determine the EC80 concentration of kinase and use this concentration of kinase in subsequent assays.

At high concentrations of kinase, a decrease in signal ratio may be observed especially with tyrosine kinases. Because the kinase itself is often phosphorylated (or can be autophosphorylated), at high concentrations, it can be bound by the Tb-labeled antibody, thereby competing with fluorescein-labeled phosphorylated substrate for binding to the antibody. If this phenomenon is observed, it indicates that a lower concentration of kinase should be used in the assay.

To determine the proper amount of kinase to use under a given set of substrate and/or ATP concentrations, you should perform the assay against a serial dilution of the kinase using a constant concentration of substrate and ATP. From the data generated, determine the EC80 (or the amount of kinase that will generate 80% of the maximal change in the FRET ratio). Use this amount of kinase for any additional optimizations. You may want to begin your kinase titration at a concentration lower than is typical in other kinase assay formats, as the LanthaScreen assay is extremely sensitive.

Typical Z' values observed are 0.7 or greater.

We have not yet developed products specific to cell-lysate based applications. However, we have shown in several cases that the Tb-based LanthaScreen signal is indeed stable in the presence of cell lysates. So, for customers building their own assays using the LanthaScreen Toolbox, the presence of cell lysate components is not expected to interfere with the LanthaScreen signal.

Terbium-labeled phosphospecific antibodies have been applied to assays for over 150 kinases. Antibodies specific to epitope tags such as 6-His or GST have been applied to nuclear receptor assays. Terbium labeled streptavidin is optimal for developing protease assays using biotinylated fluorescein labeled peptides. Ubiquitin assays for E3s have been developed using labeled ubiquitin proteins and anti-epitope tags, and deubiquitination assays can be performed using our DUB substrate. See the website (thermofisher.com/lanthascreen) for data and examples.

The LanthaScreen biochemical assay format is suitable for nearly any brand or volume of assay plate. We have developed many assays using Corning low volume black polystyrene 384 well plates (#3676). We have also successfully used Corning low volume white polystyrene 384 well plates (Cat. No. 3673), Thermo LabSystems Microfluor 1 Black 96 well plates (Cat. No. 7005), and Thermo Electron Cliniplate black polypropylene 384 well Plates (Cat. No. 95040020). When performing the assay on monochromator-based readers such as the Tecan Safire2 or the Molecular Devices M5, assay performance may be increased by using white microtiter plates. With filter-based instruments, the differences seen between black and white plates are often negligible.

We recommend the use of white assay plates for LanthaScreen GFP Cellular Assays to achieve optimal signal detection. We have successfully used Corning low-volume white polystyrene plates (Corning Cat. No. 3674) and Corning tissue culture-treated white polystyrene plates (Corning Cat. No. 3704). The tissue culture-treated plate is recommended for assay formats that perform cell treatment, lysis, and detection in the same well/plate. The low-volume plate provides excellent results in assay formats where the cell lysate and terbium-labeled detection antibody are combined in a plate different from the cell cultivation plate. We expect LanthaScreen GFP Cellular Assays to be compatible with equivalent plates from all major manufacturers.

Certain LanthaScreen GFP Cell Lines, which exhibit poor adherence to standard tissue culture-treated plates, may require the use of poly L-lysine coated plates to prevent loss of cells during medium removal.

We have found two monochromator-based instruments that work well with LanthaScreen biochemical assays; the Tecan Safire2 and the Molecular Devices SpectraMax M5. For the Safire2, we recommend exciting the samples at 332 nm with a 20 nm bandwidth. The donor (terbium) signal is measured at 485 nm with a 20 nm bandwidth and the acceptor (fluorescein) signal is measured at 515 nm with a 20 nm bandwidth. With the SpectraMax M5, we recommend exciting at 332 nm and measuring intensity at 488 and 518 nm (the band width is built in at 15 nm and cannot be adjusted).

When using a monochromator-based instrument, white assay plates often show better performance. In filter-based instruments, the color of the plate usually shows a negligible effect on data quality.

If you are using a LanthaScreen GFP Cellular Assay, we do not recommend the use of monochromator-based instruments, as the sensitivity of these instruments is not sufficient to adequately detect the endogenously expressed GFP fusion proteins.

In general, this is not possible. The typical fluorescein emission filter has a bandwidth that is too large for optimal performance in a LanthaScreen assay. Such filters not only measure the intensity of the acceptor fluorophore, but also signals from terbium itself. Having this “contaminating” intensity present in the data will make the acceptor signal only a small portion of the raw intensity. This will have a negative impact on the data.

We recommend using a 340 nm excitation filter with a 30 nm bandwidth, but an excitation filter with similar specifications will perform well. The exact specifications of the emission filters are more important. In a LanthaScreen assay, the donor (terbium) intensity is measured using a 495 nm filter (10 nm bandwidth) and the fluorescein (acceptor) intensity is measured using a 520 nm filter (25 nm bandwidth). Filters suitable for performing LanthaScreen assays can be obtained from Chroma as filter set PV001, or from other vendors. A filter module for the BMG PHERAstar can be obtained direct from BMG Instruments.

The dichroic mirror should ideally have a cutoff of less than 490 nm. A dichroic mirror meeting this specification is available from Chroma as part of filter set PV001, or from the instrument manufacturer. In some instruments (Tecan instruments, for example), the fluorescein dichroic performs well but must be manually selected. In other instruments, a 50:50 mirror may be selected, but in general will not give as high performance as a dedicated dichroic. The LanthaScreen filter module from BMG contains an optimal dichroic mirror.

The single most common reason that a TR-FRET assay fails is that an incorrect choice of emission filters was made. Unlike other fluorescent assays, the filters used in a TR-FRET assay must be exactly those that are recommended for your instrument as the emission filter choice can make or break the assay. The excitation filter has more of an impact on the assay window. Please refer to our instrument setup guides in our instrument compatibility portal (https://www.thermofisher.com/instrumentsetup). If your instrument is not listed there, please contact Drug Discovery Technical Support at drugdiscoverytech@thermofisher.com.

Note: Test your microplate reader's TR-FRET setup before you begin any work with your assay. Using reagents that you have already purchased for your assay, you can test the microplate reader's TR-FRET setup. Please refer to the Terbium (Tb) Assay (http://tools.thermofisher.com/content/sfs/manuals/LanthaScreen_Tb_Instrument_Control_Application_Note17Sep10.pdf) and Europium (Eu) Assay (http://tools.thermofisher.com/content/sfs/manuals/Eu_Instrument_Control_AppNote_12Nov10.pdf) Application Notes for plate reader setup.