IMAP-FP-Kinase Assay Using the SpectraMax M5 Multiplate Reader (2023)

Protein kinases play a central role in the regulation of many cellular processes. In recent years, they have become one of the most important targets for drugs against cancer and many other diseases. IMAP^®Molecular Devices' technology enables rapid, non-radioactive testing of multiple kinases and is suitable for assay development and high-throughput screening. The IMAP technology is based on the combination of immobilized metal coordination complexes and phosphates on nanoparticles. When the IMAP-binding moiety binds to the phosphopeptide generated by the kinase reaction, the molecular motion of the peptide is altered and the fluorescence polarization of the peptide-bound fluorescent label increases (Figure 1). This homogeneous assay is easy to perform and is applicable to many kinases regardless of peptide substrate sequence

Figure 1. Principle of IMAP FP assay.IMAP-FP Kinase Assay Chart

Spectrum max^®The M5 Multimode Microplate Reader is ideal for assay development and high-throughput screeningIMAP test. This monochromator-based instrument allows the user to select the optimal wavelength for each selected fluorophore without the need to purchase additional filter sets. This application note describes how to perform an IMAP fluorescence polarization (FP) kinase assay and calibration curve using green and red labeled fluorogenic substratesSpectrum Max M5Microplate Readers and SoftMax^®Professional software. Generated for Lck, a tyrosine kinase that plays a key role in T cell signaling, and Akt1/PKBα, a serine-threonine kinase involved in phosphatidylinositol 3-kinase signaling and cell survival Enzyme dilution curve. The inhibitory effect of staurosporine on Akt1/PKBα was also tested. High Z'-factor values ​​were obtained with both FAM and TAMRA-labeled peptide substrates, data comparable to filter-based analysts^®HT Multimode Microplatten Reader.

Material

• Progressive Binding System IMAP Screening Express Kit (Molecular Devices, catalog # R8125 )
  • Progressive IMAP Binding Reagent
  • IMAP Progressive Binding Buffer A (5X)
  • IMAP progressive binding buffer B (5X)
  • IMAP response buffer (5X)
• Lck-Kinase (Upstate Cat. Nr. 14-442) Kat.-Nr. #R7159)
  • FAM-p34cdc2-derived peptide (Molecules Devices cat# R7157)
  • TAMRA-p34cdc2 Derived Peptide (Molecular Devices Cat# R7309)
  • FAM-p34cdc2 Derived Phosphopeptide Calibrator (Molecular Devices Cat# R7271)
• Akt1-PKBα-Kinase (Northern Cat# 14-276)
  • FAM-Crosstide（Molecular Devices Kat. Nr. R7110）
  • FAM-Phospho-Crosstid（Molecular Devices Kat. Nr. R7159）
• Adenosine 5'-triphosphate (ATP), 50 mM purified water stock solution (Sigma Cat# A6559)
• DL-Dithiothreitol (DTT), 100 mM purified water stock solution (Sigma Cat.#D9779)
• Staurosporine (Biomol Cat. Nr. EI-156)
• 384-well black polystyrene microplates (Corning Cat. #3710)
• Spectrum Max M5Multi-mode microplate readerSoftMax Pro software(molecular devices)

method

Kinase reaction

Step 1: Prepare Complete Reaction Buffer (CRB) by adding DTT to a final concentration of 1mM (1:100 dilution of 100mM DTT stock solution) in 1X IMAP Reaction Buffer.

Step 2: Make a 400 nM (4X) stock solution of FAMor TAMRA-labeled peptide substrate in CRB (1:50 dilution of 20 µM peptide stock).

Step 3: Prepare 20 µM (4X) ATP stock in CRB (1:2500 dilution of 50 mM ATP stock).

Step 4: Prepare an enzyme dilution series at 4x the final concentration required for the assay. For kinase inhibition assays, use a constant enzyme concentration and prepare serial dilutions of staurosporine or other kinase inhibitors. Use CRB for dilution.

Step 5: Pipette the following into the quadruple enzyme assay wells to set up the kinase reaction:

• 5 µL CRB or staurosporine
• 5 µL Enzyme (for background samples without enzymes, replace 5 µL CRB)
• 5 µL of 20 µM ATP stock solution
• 5 ml 400-nM-Peptidstamm

Step 6: Incubate at room temperature for 1 to 1.5 hours.

Note: For more detailed instructions, see your IMAP product manual.

calibration standard

Step 1: Prepare a 100 nM peptide stock solution in CRB (using the same peptide substrate as in the kinase assay).

Step 2: Prepare a 100 nM phosphopeptide stock solution in CRB (using the phosphorylated version of the substrate used in the kinase assay).

Step 3: Combine peptide and phosphopeptide stocks to prepare calibration standards, as shown in Table 1. The amounts given are sufficient to prepare quadruple samples.

Table 1. Preparation of calibration standards.

Step 4: Pipette 20 µL of each calibration standard into four wells, including one set of pure buffer background samples containing 20 µL of CRB.

binding reaction

Step 1: Prepare Progressive Binding Buffer by mixing 75% Progressive Binding Buffer A with 25% Progressive Binding Buffer B.

Step 2: Prepare the binding solution by diluting the progressive binding reagent 1:600 ​​in the progressive binding buffer.

Step 3: Pipette 60 µL of binding solution into each test and calibration standard well (including buffered background samples).

Step 4: Incubate at room temperature for 1 hour in the dark.

To set up templates in SoftMax Pro and read plates on a SpectraMax M5 plate reader:

Note: IMAP FP protocols for FAM and TAMRA labeled substrates can be found in the Binding Assays protocol folder of SoftMax Pro 5.

Step 1: Open the SoftMax Pro IMAP FP log specific to the fluorophore used. FAM and TAMRA protocols are available; adjust wavelength settings if using fluorophores other than FAM or TAMRA. Set asSpectrum Max M5Microplate readers are listed in Table 2.

Table 2. IMAP FP settings for the SpectraMax M5 plate reader in SoftMax Pro

Step 2: Set up the experiment template, where background samples, kinase assay samples, and calibration standards are specified. Kinase assay samples and calibration standards can be assigned to eight preconfigured "sample" groups in the SoftMax Pro template. Assigning the buffer-only control to the appropriate "background" group allows automatic subtraction of background fluorescence prior to calculating millipolarization (mP) values.

Step 3: Place the microplate in the plate reader tray - make sure to include the purple plate adapter as the plate is read from above - and click "Read".

result

The SoftMax Pro IMAP FP protocol automatically calculates mean parallel and perpendicular values, mP, total intensity, standard deviation and CV. In the group table for each test sample, calculate the mP subtracted from the background using the background sample given in the template as a reference. Percent phosphorylation can also be calculated by extrapolation from a suitable calibration curve.

Figure 2 shows the Lck kinase dilution curve obtained using FAM-p34cdc2 derived peptides. When the plate was read on a SpectraMax M5 microplate reader, the enzyme concentration range was 0.04 to 3.5 units/mL, the Δ mP was 303, and the Z' factor was 0.87.²Figure 3 shows the same test panel read on an Analyst HT multimode reader. Here the delta mP is 336 and the Z' factor is 0.95. On both tools, the EC₅₀0.5 units/ml.

Figure 2. IMAP-FP Kinase Assay on SpectraMax M5 Microplate Reader.Lck kinase dilution curves of FAM-p34cdc2-derived peptides from 0.04 to 3.5 units/ml enzyme read on a SpectraMax M5 (4-parameter curve fitting). Error bars here and in all subsequent graphs are standard deviations.

Figure 3. IMAP-FP Kinase Assay on Analyst HT.Lck kinase dilution curves from 0.04 to 3.5 units/mL enzyme using FAM-p34cdc2 derived peptides, read on Analyst HT (4 parameter curve fitting).

If you run separate calibration curves with unphosphorylated and phosphorylated peptide controls, mP values ​​can be converted to percent phosphorylation (Figure 4). First, create a new graph ("Plot #1" is the name assigned by default by the software) and plot mP versus percent phosphorylation (concentration) for the calibrator sample set. The default name of the plot is "Plot#1". In the Calibration Standard Sets table, insert a new column with the following formula: InterpX('Plot#1@Graph#1@IMAP FP_FAM',AvgbkgsubmP). "IMAP FP_FAM" is the test header.

Figure 4. IMAP with FAM-tagged peptides.Calibration standard curves of FAM-p34cdc2-derived peptides and phosphopeptides read on a SpectraMax M5 (square curve fit).

To determine the performance of the red fluorophore IMAP kinase assay, another Lck kinase dilution curve was constructed using TAMRA-p34cdc2-derived peptides (Figure 5). Here, a Δ mP of 258 and a Z' factor of 0.95 were measured on a SpectraMax M5 microplate reader over an enzyme concentration range of 0.04 to 3.5 units/mL. In Figure 6, the same test panel was read on an Analyst HT reader with a delta mP of 270 and a Z' factor of 0.93. On both tools, the EC₅₀is 0.4 units/ml, which is close to the EC value₅₀Substrates with the FAM mark. A corresponding calibration curve was also generated (Figure 7). Results obtained with TAMRA-labeled peptide substrates were similar to those obtained with FAM-labeled substrates on both instruments, enabling users to utilize red-labeled fluorophores, including minimizing background fluorescence from test compounds.

Figure 5. Lck kinase assay using TAMRA-labeled peptides on a SpectraMax M5 microplate reader.Lck kinase dilution curves of TAMRA-p34cdc2-derived peptides were read on a SpectraMax M5 (4-parameter curve fitting).

Figure 6. Lck kinase assay using TAMRA-labeled peptides on the Analyst HT.Dilution curves of Lck kinase with TAMRA-p34cdc2-derived peptides, please read on Analyst HT (4-parameter curve fitting).

Figure 7. IMAP with TAMRA-tagged peptides.Calibration standard curves (linear curve fitting) of TAMRA-p34cdc2-derived peptides and phosphopeptides read on a SpectraMax M5.

To demonstrate the performance of the IMAP FP assay using different kinases on a SpectraMax M5 microplate reader, an enzyme dilution curve of Akt1/PKBα was generated using FAM-Crosstide as substrate. In Figure 8, an enzyme concentration range of 0.002 to 5.4 units/mL yielded a delta mP of 339 and a Z' factor of 0.92. Inhibition of Akt1/PKBα by staurosporine is shown in Figure 9; here the delta mP is 199 and the Z' factor is 0.93. Calculated IC₅₀The concentration of staurosporine in this test was 19.7 nM. The delta mP here is lower than the value in the enzyme dilution curve because the kinase concentration chosen for the inhibition assay resulted in a maximal phosphorylation of approximately 75%.

Figure 8. Akt/PBKa kinase assay using FAM-labeled peptides on a SpectraMax M5 plate reader.Akt1/PKBα kinase dilution curves using FAM Crosstide substrate were read on a SpectraMax M5 (4-parameter curve fitting).

Figure 9. Kinase inhibition on the SpectraMax M5 plate reader.Akt1/PKBα kinase inhibition curves for staurosporine and FAM Crosstide substrates were read on a SpectraMax M5. The five-parameter curve fitting available in SoftMax Pro 5 is used here.

diploma

As researchers seek to understand the role of kinases in cell signaling and disease, and to identify modulators of kinases that could be used as therapeutics, they are increasingly looking for non-radioactive, Homogeneous assay method. Homogeneous FP assays provide an HTS-friendly format for the identification of kinase activators, inhibitors, and substrates. IMAP-FP kinase assay inSpectrum Max M5Microplate readers provide accurate, reproducible data with reliable z'-factor values. The dual monochromator system allows users to optimize assays using a variety of fluorescently labeled peptide substrates without the need to purchase additional filter sets. Results from IMAP-FP kinase assays using green or red labeled substrates were very similar to those obtained using filter-based Analyst microplate readers. The IMAP-FP protocol in SoftMax Pro 5 provides a convenient way to acquire FP data and perform calculations, and can be easily adapted to any fluorophore.

reference

1. Product leaflet for IMAP Akt Assay Kit (Molecules Devices product #R8058).
2. Zhang Jianhua, Zhong Deyi and Oldenburg, K.R. (1999). Simple statistical parameters for evaluating and validating high-throughput screening assays.Journal of Biomolecular Screening4（2）：67-73。

Learn more about IMAP detection >>

Protein kinases play a central role in the regulation of many cellular processes. In recent years, it has become an important drug target for cancer and many other diseases. IMAP is a rapid, non-radioactive kinase assay from Molecular Devices. Due to the inherent characteristics of the technology, it is very suitable for test optimization and high-throughput screening. The principle of IMAP detection technology is that phosphate ions are combined with nanosphere particles with trivalent metal ions immobilized on the surface. When this binding reaction occurs, the trajectory of the fluorescently labeled polypeptide molecule changes, and the fluorescence polarization of the fluorescently labeled complex increases (see Figure 1). Because it is a homogeneous assay that ignores the sequence of the substrate polypeptide, it is often used to detect different kinases.

Figure 1. Principle of IMAP FP assay.IMAP-FP Kinase Assay Chart

The SpectraMax M5 MultiMode Reader is the ideal and reliable detection platform when using IMAP technology for assay optimization and high-throughput screening. SpectraMax M5 is a multifunctional microplate reader based on a grating monochromator. Users can freely choose different detection wavelengths for different fluorescent dye molecules without purchasing additional filter accessories. This application note details how to use the SpectraMax M5 Multiplate Reader and SoftMax Pro Software to detect and calibrate curves for red and green fluorescent substrates when performing an IMAP fluorescent polarized kinase assay. LCK is a tyrosinase that plays a key role in the T cell signal transduction pathway, and its dilution curve was constructed. In addition, Akt1/PKBa, a serine-threonine kinase, is involved in phosphatidylinositol kinase 3 signaling and cell viability. The Z'-factor values ​​obtained at the beginning of the reaction process were calculated separately for the FAM and TAMRA-labeled peptide substrates in experiments in which staurosporine inhibited Akt1/PKBa, and the results were consistent with those obtained on the multiplex filter type - Functional The results of the microplate reader were consistent.

Material

• IMAP-Schnell Screening Kit (Molecular Devices, Kat.-Nr. R8125)
  • IMAP Binding Reagents
  • IMAP binding buffer A (5X)
  • IMAP binding buffer B (5X)
  • IMAP response buffer (5X)
• LCK-Kinase (Northern Cat. No. 14-442)
  • Markiertes FAM-p34cdc2-Peptidsubstrat（Molecular Devices Kat.-Nr. R7157）
  • Markiertes TAMRA-p34cdc2-Peptidsubstrat（Molecular Devices Kat.-Nr. R7309）
  • Labeled phosphopeptide for FAM-p34cdc2 Starting Calibration Reagent (Molecules Devices Cat# R7271)
• Akt1-PKBa-Kinase (Northern Cat. No. 14-276)
  • FAM-markiertes Peptidsubstrat（Molecular Devices Kat.-Nr. R7110）
  • FAM-tagged phosphorylated peptide substrate (Molecular Devices catalog #R7159)
• 50mM Adenosine 5'-triphosphate (ATP) in purified water (Sigma Cat. #A6559)
• 100mM DL-Dithiothreitol (DTT) in purified water (Sigma Cat. #D9779)
• Staurosporin Biomol Cat #EI-156)
• 384-well black polystyrene plate (Corning Cat. #3710)
• The computer was preloaded with SoftMax Pro software and connected to a SpectraMax M5 multi-function microplate reader (Molecular Devices).

method

Kinase reaction

Step 1. Add DTT to 1xIMAP Reaction Buffer and make sure the final concentration of DTT is 1mM (1:100 dilution of 100mM DTT stock solution) to obtain Complete Reaction Buffer (CRB).

Step 2. Prepare 400 nM (4x) FAM or TAMRA-labeled peptide substrate in CRB (1:50 dilution of original 20 µM peptide stock);

Step 3. Prepare 20 μM (4x) ATP in CRB (1:2500 dilution of original 50 mM ATP stock solution);

Step 4. Prepare serial dilutions of the enzyme to a final concentration of 4x. For kinase inhibitor assays, use a constant enzyme concentration and prepare a series of serial dilutions of staurosporine or other kinase inhibitors.

Step 5. Add the following reagents in parallel to 4 replicate wells of the Kinase Reaction System:

• 5ul CRB or staurosporine
• 5 µl enzyme (or 5 µl CRB for background wells without enzyme added)
• 5 µl 20 µM ATP stock solution
• 5ul 400nM peptide stock solution

Step 6. Incubate at room temperature for 1 to 1.5 hours.

Note: A more detailed description can be found in IMAP Product Insert 1. In addition, the experimental data of various kinases can also be browsed at the following websitehttp://www.moleculardevices.com/assayarChinese chives/

Preparation of Calibration Standards

Step 1. Prepare a 100 nM peptide stock in CRB (using the same substrate as in the kinase assay);

Step 2. Prepare a 100 nM phosphopeptide stock solution in CRB (using the same phosphorylated substrate as in the kinase assay);

Step 3. As shown in Table 1, after mixing the polypeptide stock solution and the phosphorylated polypeptide according to certain rules, a calibration standard was prepared. The sample volume is sufficient for 4 replicate wells;

Note: For more information on preparing and using calibration curves, see IMAP Application Note #4, Developing IMAP2 Calibration Curves.

Step 4. In each of 4 replicate wells, draw 20 µl of calibration standards, including a set of blank background samples containing only 20 µl of CRB.

binding reaction

Step 1. Mix 75% Binding Buffer A and 25% Binding Buffer B to make Binding Buffer;

Step 2: Prepare the binding solution Step 2. Dilute the binding reagent 1:600 ​​to form a binding buffer to obtain a binding reaction solution.

Step 3. Pipette 60 µL of Binding Reaction Solution into each assay well and calibration standard well (including background well samples).

Step 4. Incubate at room temperature in the dark for 1 hour;

To set up templates in SoftMax Pro and read plates on a SpectraMax M5 plate reader:

Note: IMAP FP protocols for FAM and TAMRA labeled substrates can be found in the Binding Assays protocol folder of SoftMax Pro 5.

Step 1: Open the SoftMax Pro IMAP FP log specific to the fluorophore used. FAM and TAMRA protocols are available; adjust wavelength settings if using fluorophores other than FAM or TAMRA. Set asSpectrum Max M5Microplate readers are listed in Table 2.

Table 2. IMAP FP settings for the SpectraMax M5 plate reader in SoftMax Pro

Step 2: Set up the test template, select background sample wells, kinase sample wells and calibration standard wells. Kinase assay samples and calibration standards can be grouped into 8 preset sample groups in the SoftMax Pro software template. Pure buffered controls were first separated into appropriate background groups and their background fluorescence values ​​were automatically subtracted using the software before calculating minimum polarization (mP) values.

Step 3. After inserting the plate into the plate reader - make sure the purple holder (adapter) is placed on the plate holder - then click "Read" to read the plate.

result

The preset IMAP fluorescence polarization templates in SoftMax Pro software automatically calculate the mean horizontal and vertical polarization (mP values), total fluorescence intensity, standard deviation, and CV values. In the group column of each test sample, the template automatically subtracts the fluorescence polarization value of the background sample, and then calculates the minimum mP polarization value. Phosphorylation ratios of samples can also be calculated from the corresponding calibration curves. (See IMAP Application Note #4, "Development of an IMAP Calibration Curve")

Figure 2 shows the dilution curve of LCK kinase obtained using the FAM-p34cdc2 peptide. Enzyme concentrations ranging from 0.04 to 3.5 units/mL were obtained using a SpectraMax M5 microplate reader with a minimum polarization value of 303 and a Z' factor value of 0.87. Figure 3 shows that when the same well was read using an Analyst HT Multi-Read microplate reader, the fluorescence polarization value was 336 and the Z' factor was 0.95. EC50 values ​​were determined to be 0.5 units/ml for both devices.

Figure 2. IMAP-FP Kinase Assay on SpectraMax M5 Microplate Reader.Lck kinase dilution curves of FAM-p34cdc2-derived peptides from 0.04 to 3.5 units/ml enzyme read on a SpectraMax M5 (4-parameter curve fitting). Error bars here and in all subsequent graphs are standard deviations.

Figure 3. IMAP-FP Kinase Assay on Analyst HT.Lck kinase dilution curves from 0.04 to 3.5 units/mL enzyme using FAM-p34cdc2 derived peptides, read on Analyst HT (4 parameter curve fitting).

When using non-phosphorylated and phosphorylated peptide samples as quality controls to construct a calibration curve, fluorescence polarization values ​​can be displayed as a percentage to determine the degree of phosphorylation. (See Figure 4) First, create a new curve (the default name of the software is Chart #1), and use the fluorescence polarization value (mP value) relative to the phosphorylation percentage (concentration) as the quality control sample group to draw the curve. The default plot name is "Plot#1". In the Calibration Standard Set column, create a new column and enter the following formula, namely: InterpX('Plot#1@Graph#1@IMAP FP_FAM',AvgbkgsubmP). And use "IMAP FP_FAM" as the test name.

Figure 4. IMAP with FAM-tagged peptides.Calibration standard curves of FAM-p34cdc2-derived peptides and phosphopeptides read on a SpectraMax M5 (square curve fit).

To determine the effect of the IMAP kinase assay using the red fluorescent dye, another LCK kinase dilution curve result was obtained for the TAMRA p34cdc2 polypeptide. (See Figure 5.) Results were recorded using a SpectraMax M5 microplate reader. Enzyme concentrations ranged from 0.04 to 3.5 units/ml, fluorescence polarization (mP) values ​​were 258, and Z'-factor values ​​were 0.95. In Figure 6, the same assay was performed on an Analyst HT microplate reader, resulting in a fluorescence polarization (mP value) of 270 and a Z' factor of 0.93. Both readers gave an EC50 value of 0.4 units/mL, consistent with the results for the FAM-labeled substrate. A corresponding calibration curve was also generated (see Figure 7). Similar results for TAMRA and FAM also allow users to take advantage of red fluorescent dyes, which minimize background fluorescence of compounds.

Figure 5. Lck kinase assay using TAMRA-labeled peptides on a SpectraMax M5 microplate reader.Lck kinase dilution curves of TAMRA-p34cdc2-derived peptides were read on a SpectraMax M5 (4-parameter curve fitting).

Figure 6. Lck kinase assay using TAMRA-labeled peptides on the Analyst HT.Dilution curves of Lck kinase with TAMRA-p34cdc2-derived peptides, please read on Analyst HT (4-parameter curve fitting).

Figure 7. IMAP with TAMRA-tagged peptides.Calibration standard curves (linear curve fitting) of TAMRA-p34cdc2-derived peptides and phosphopeptides read on a SpectraMax M5.

Figure 8. Akt/PBKa kinase assay using FAM-labeled peptides on a SpectraMax M5 plate reader.Akt1/PKBα kinase dilution curves using FAM Crosstide substrate were read on a SpectraMax M5 (4-parameter curve fitting).

Figure 9. Kinase inhibition on the SpectraMax M5 plate reader.Akt1/PKBα kinase inhibition curves for staurosporine and FAM Crosstide substrates were read on a SpectraMax M5. The five-parameter curve fitting available in SoftMax Pro 5 is used here.

Summarize

As researchers strive to understand the role of kinases in cell signaling pathways and various diseases, as well as the regulatory mechanisms they exert in the treatment of many diseases, they increasingly desire non-radioactive, homogeneous assays that can be used for assay optimization and high-level testing. - Throughput drug screening. Fluorescence polarization assays provide a homogenous, high-throughput method for identifying activators, inhibitors, and substrates of various kinases. IMAP fluorescence polarization detection using SpectraMax M5 multi-function microplate reader: The obtained results showed high precision, good reproducibility and high Z' factor value. The dual-gate monochromator system allows the user to select peptides labeled with different fluorochromes on the substrate for optimized assays without the need for additional optional filters. In addition, the results obtained with fluorescent polarized kinase IMAP assays using substrates labeled with green or red fluorescent dyes were consistent with those obtained with microplate readers using filter-type monochromators. Finally, the preset IMAP fluorescence polarization templates in SoftMax Pro software provide a more convenient way to acquire and analyze fluorescence polarization data, and these templates can also be used for the detection of various fluorescent dye molecules.

reference

1. Product leaflet for IMAP Akt Assay Kit (Molecules Devices product #R8058).
2. Zhang Jianhua, Zhong Deyi and Oldenburg, K.R. (1999). Simple statistical parameters for evaluating and validating high-throughput screening assays.Journal of Biomolecular Screening4（2）：67-73。