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Protein kinases are central to the regulation of many cellular processes. In recent years they have emerged as one of the most important classes of drug targets for cancer and many other diseases. IMAP^® Technology from Molecular Devices enables rapid, non-radioactive assay of a wide array of kinases and is suited to both assay development and high-throughput screening. IMAP technology is based on binding of phosphate through immobilized metal coordination complexes on nanoparticles. When IMAP binding entities bind to phosphopeptides generated by a kinase reaction, molecular motion of the peptide is altered and fluorescence polarization for the fluorescent label attached to the peptide increases (Figure 1). This homogeneous assay is easy to perform and applicable to many kinases regardless of peptide substrate sequence

Figure 1. IMAP FP assay principle. Diagram of the IMAP FP kinase assay

The SpectraMax^® M5 Multi-Mode Microplate Reader is ideally suited for assay development and high-throughput screening using IMAP assays. This monochromator-based instrument allows users to select optimal wavelengths for any chosen fluorophore without the need to purchase additional filter sets. This application note describes how to run IMAP fluorescence polarization (FP) kinase assays and calibration curves with green- and red-labeled fluorescent substrates using the SpectraMax M5 microplate reader and SoftMax^® Pro Software. Enzyme dilution curves were performed for Lck, a tyrosine kinase with a key role in T-cell signaling, and Akt1/ PKBα, a serine-threonine kinase involved in phosphatidylinositol 3-kinase signaling and cell survival. Inhibition of Akt1/PKBα by staurosporine was also assayed. High Z’ factor values are obtained with both FAMand TAMRA-labeled peptide substrates, with data comparable to those obtained on the filter-based Analyst^® HT Multi-Mode Microplate Reader.

Materials

• IMAP Screening Express Kit with Progressive Binding System (Molecular Devices Cat. #R8125)
  • IMAP Progressive Binding Reagent
  • IMAP Progressive Binding Buffer A (5X)
  • IMAP Progressive Binding Buffer B (5X)
  • IMAP Reaction Buffer (5X)
• Lck kinase (Upstate Cat. #14-442) Cat. #R7159)
  • FAM-p34cdc2-derived peptide (Molecular Devices Cat. #R7157)
  • TAMRA-p34cdc2-derived peptide (Molecular Devices Cat. #R7309)
  • FAM-p34cdc2-derived phosphopeptide calibrator (Molecular Devices Cat. #R7271)
• Akt1-PKBα kinase (Upstate Cat. #14-276)
  • FAM-Crosstide (Molecular Devices Cat. #R7110)
  • FAM-phospho-Crosstide (Molecular Devices Cat. #R7159)
• Adenosine 5’ triphosphate (ATP), 50 mM stock in purified water (Sigma Cat. #A6559)
• DL-Dithiothreitol (DTT), 100 mM stock in purified water (Sigma Cat. #D9779)
• Staurosporine (Biomol Cat. #EI-156)
• Black 384-well polystyrene microplate (Corning Cat. #3710)
• SpectraMax M5 Multi-Mode Microplate Reader with SoftMax Pro Software (Molecular Devices)

Methods

Kinase reactions

Step 1: Prepare Complete Reaction Buffer (CRB) by supplementing 1X IMAP Reaction Buffer with DTT at a final concentration of 1 mM (1:100 dilution of 100 mM DTT stock).

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

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

Step 4: Prepare an enzyme dilution series at 4X final desired concentrations for the assay. For the kinase inhibition assay, use a constant concentration of enzyme and make a dilution series of staurosporine or other kinase inhibitor. Use CRB for dilutions.

Step 5: Set up kinase reactions by pipetting the following into quadruplicate enzyme assay wells:

• 5 µL CRB or staurosporine
• 5 µL enzyme (for no-enzyme background samples, substitute 5 µL CRB)
• 5 µL of 20-µM ATP stock
• 5 mL of 400-nM peptide stock

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

Note: For more detailed instructions, please refer to the IMAP product insert.

Calibration standards

Step 1: Make a 100-nM peptide stock in CRB (use the same peptide substrate as in the kinase assay).

Step 2: Make a 100-nM phosphopeptide stock in CRB (use the phosphorylated version of the substrate used in the kinase assay).

Step 3: Combine peptide and phosphopeptide stocks to make calibration standards as indicated in Table 1. Amounts given are sufficient to set up quadruplicate samples.

Table 1. Preparation of calibration standards.

Step 4: Pipet 20 µL each calibration standard into quadruplicate wells, including a set of buffer-only background samples containing 20 µL CRB.

Binding reaction

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

Step 2: Prepare Binding Solution by diluting Progressive Binding Reagent 1:600 in Progressive Binding Buffer.

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

Step 4: Incubate at room temperature for 1 hour, protected from light.

Set up template in SoftMax Pro and read plate on the SpectraMax M5 microplate reader:

Note: IMAP FP protocols for use with FAM- and TAMRA-labeled substrates are available in SoftMax Pro 5 in the Binding Assays protocol folder.

Step 1: Open the SoftMax Pro IMAP FP protocol specific to the fluorophore used. FAM and TAMRA protocols are available; adjust wavelength settings if a fluorophore other than FAM or TAMRA is used. Settings for the SpectraMax M5 microplate reader are shown in Table 2.

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

Step 2: Set up an experiment template designating background samples, kinase assay samples, and calibration standards. Kinase assay samples and calibration standards may be assigned to eight preconfigured ‘Sample’ groups in the SoftMax Pro template. Assigning buffer-only controls to the corresponding ‘Background’ groups enables automatic subtraction of backgroundfluorescence prior to calculation of milli-polarization (mP) values.

Step 3: Place the microplate in the plate reader carriage—be sure to include a purple plate adapter since the plate is read from the top—and click Read.

Results

The SoftMax Pro IMAP FP protocol automatically calculates average parallel and perpendicular values, mP, total intensity, standard deviations, and CV. In the group table for each set of assay samples, background subtracted mP is calculated using background samples designated in the template as references. Percent phosphorylation can also be calculated by extrapolating from an appropriate calibration curve.

Figure 2 shows a Lck kinase dilution curve obtained using the FAM-p34cdc2-derived peptide. When the plate was read on the SpectraMax M5 microplate reader, an enzyme concentration range of 0.04 to 3.5 units/mL yielded a delta mP of 303, with a Z’ factor of 0.87.² Figure 3 shows the same assay plate read on the Analyst HT multi-mode reader. Here the delta mP was 336, and Z’ factor was 0.95. On both instruments the EC₅₀ was 0.5 units/mL.

Figure 2. IMAP FP kinase assay on SpectraMax M5 microplate reader. Lck kinase dilution curve with FAM-p34cdc2-derived peptide from 0.04 to 3.5 units/mL enzyme, read on SpectraMax M5 (4-parameter curve fit). Error bars here and on all subsequent graphs are standard deviations.

Figure 3. IMAP FP kinase assay on Analyst HT. Lck kinase dilution curve with FAM-p34cdc2-derived peptide from 0.04 to 3.5 units/mL enzyme, read on Analyst HT (4-parameter curve fit).

Values for mP may be converted to percent phosphorylation if you set up a separate calibration curve using nonphosphorylated and phosphorylated peptide controls (Figure 4). First create a new graph (‘Graph#1’ is the default name assigned by the software) and plot mP vs. percent phosphorylated (concentration) for your calibrator sample group. The default name for this plot is ‘Plot#1.’ In the calibration standards group table, insert a new column with the following formula: InterpX(‘Plot#1@Graph#1@IMAP FP_FAM’,AvgbkgsubmP). ‘IMAP FP_FAM’ is the experiment title.

Figure 4. IMAP with FAM-labeled peptide. Calibration standard curve with FAM-p34cdc2-derived peptide and phosphopeptide, read on SpectraMax M5 (quadratic curve fit).

To determine performance of the IMAP kinase assay with red fluorophore, another Lck kinase dilution curve was performed using the TAMRA-p34cdc2- derived peptide (Figure 5). Here, an enzyme concentration range of 0.04 to 3.5 units/mL yielded a delta mP of 258, with a Z’ factor of 0.95 when read on the SpectraMax M5 microplate reader. In Figure 6, the same assay plate was read on the Analyst HT reader, with a delta mP of 270 and Z’ factor of 0.93. On both instruments the EC₅₀ was 0.4 units/mL, a value close to the EC₅₀ with FAM-labeled substrate. A corresponding calibration curve was also set up (Figure 7). Similarity of results with TAMRA-labeled peptide substrate on both instruments to those obtained with FAM-labeled substrate allow users to take advantage of the benefits of red-labeled fluorophores, including minimizing background fluorescence emitted by test compounds.

Figure 5. Lck kinase assay with TAMRA-labeled peptide on SpectraMax M5 microplate reader. Lck kinase dilution curve with TAMRA-p34cdc2-derived peptide, read on SpectraMax M5 (4-parameter curve fit).

Figure 6. Lck kinase assay with TAMRA-labeled peptide on Analyst HT. Lck kinase dilution curve with TAMRA-p34cdc2-derived peptide, read on Analyst HT (4-parameter curve fit).

Figure 7. IMAP with TAMRA-labeled peptide. Calibration standard curve with TAMRA-p34cdc2- derived peptide and phosphopeptide, read on SpectraMax M5 (linear curve fit).

To demonstrate performance of the IMAP FP assay on the SpectraMax M5 microplate reader with another kinase, an enzyme dilution curve for Akt1/PKBα was obtained using FAM-Crosstide as a 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 was 199 with a Z’ factor of 0.93. The calculated IC₅₀ for staurosporine for this assay was 19.7 nM. The delta mP was lower here than in the enzyme dilution curves because a concentration of kinase was chosen for the inhibition assay that gave about 75% maximal phosphorylation.

Figure 8. Akt/PBKa kinase assay with FAM-labeled peptide on SpectraMax M5 microplate reader. Akt1/PKBα kinase dilution curve with FAM-Crosstide substrate, read on SpectraMax M5 (4-parameter curve fit).

Figure 9. Kinase inhibition on SpectraMax M5 microplate reader. Akt1/PKBα kinase inhibition curve with staurosporine and FAM-Crosstide substrate, read on SpectraMax M5. A five-parameter curve fit, available in SoftMax Pro 5, was used here.

Conclusion

As researchers seek to understand the roles that kinases play in cell signaling and disease, as well as identify kinase modulators that may serve as therapeutics, they are increasingly looking to nonradioactive, homogenous assays that are well suited to both assay development and high-throughput screening. Homogeneous FP assays provide an HTS-friendly format for identifying kinase activators, inhibitors, and substrates. IMAP FP kinase assays run on the SpectraMax M5 microplate reader yield precise, reproducible data with robust Z’ factor values. The dual monochromator system allows users to optimize the assays using a wide range of fluorescently labeled peptide substrates without the need to purchase additional filter sets. Results for IMAP FP kinase assays with either green- or red-labeled substrates are very similar to results obtained with the filterbased Analyst microplate readers. IMAP FP protocols in SoftMax Pro 5 provide a convenient way to obtain and perform calculations on FP data and are easily adaptable to any fluorophore.

References

1. IMAP Akt Assay Kit Product Insert (Molecular Devices product #R8058).
2. Zhang, J.H., Chung, T.D.Y., and Oldenburg, K.R. (1999). A simple statistical parameter for use in evaluation and validation of highthroughput screening assays. J Biomol Screen 4(2): 67-73.

Learn more about IMAP Assays >>

蛋白激酶在调节许多细胞反应过程时起着核 心的作用。近年来已经成为癌症和其它许多 疾病药物重要的作用靶点。IMAP是Molecular Devices 公司推出一种快速、非放射性的 激酶检测技术，由于技术本身特点其非常适 合 用 于 进 行 试 验 优 化 和 高 通 量 的 筛 选 。 IMAP检测技术基于的原理是磷酸根离子与 表面固化有三价金属离子的纳米球颗粒的一 种结合作用。当这种结合反应发生后，荧光 标记的多肽分子的运动轨迹发生了改变，荧 光标记复合物的荧光偏振值就会增加 (SeeFigure 1.)。因为是一种均相的，可忽 视底物多肽序列的一种检测方法，所以可广 泛的应用于多种激酶的检测。

Figure 1. IMAP FP assay principle. Diagram of the IMAP FP kinase assay

当使用 IMAP技术进行试验优化和高通量的 筛选时，SpectraMax M5多功能微孔板读板 机可作为其非常理想的、可靠的检测平台。 SpectraMax M5是一种基于光栅型单色器 的多功能微孔板读板机，允许使用者针对不 同的荧光染料分子随意选择不同的检测波 长，而无需额外再选购滤光片配件。这篇应 用文章详细的描述了在进行IMAP荧光偏振 激酶试验时，如何使用SpectraMax M5 多 功能微孔板读板机和 SoftMax Pro 软件进 行检测和分别对红色及绿色荧光底物来校正 曲线。LCK是一种酪氨酸酶，在T细胞信号 转导通路中发挥着非常关键作用，制备其 稀释曲线。另外Akt1/PKBa，一种丝氨酸苏氨酸激酶，参与磷脂酰肌醇激酶3的信 号转导和细胞活力。通过十字孢碱抑制 Akt1/PKBa的试验中，计算FAM和TAMRA 分别标记的多肽底物在反应过程始中获得 的Z'因子值，其结果与滤光片式多功能微孔 板读板机上获得的结果相一致。

材料

• IMAP 快速筛选试剂盒 ( Molecular Devices Cat. #R8125)
  • IMAP结合试剂
  • IMAP 结合缓冲液 A (5X)
  • IMAP结合缓冲液B (5X)
  • IMAP反应缓冲液（5X）
• LCK激酶(Upstate Cat. #14-442)
  • 标记 FAM-p34cdc2多肽底物 (Molecular Devices Cat. #R7157)
  • 标记 TAMRA-p34cdc2 多肽底物 (Molecular Devices Cat. #R7309)
  • 标记FAM-p34cdc2来源的磷酸化多肽 校正试剂(Molecular Devices Cat. #R7271)
• Akt1-PKBa 激酶 (Upstate Cat. #14-276)
  • FAM标记多肽底物 (Molecular Devices Cat.#R7110)
  • FAM标记的磷酸化多肽底物 (Molecular Devices Cat. #R7159)
• 贮存在纯水中的50 mM Adenosine 5’ triphosphate (ATP)(Sigma Cat. #A6559)
• 贮存在纯水中的100 mM DL-Dithiothreitol (DTT)(Sigma Cat. #D9779)
• 十字孢碱 (Staurosporine) Biomol Cat. #EI-156)
• 黑色聚苯乙烯材质的384孔板(Corning Cat. #3710)
• 预装有SoftMax Pro软件电脑及连接 SpectraMax M5 多功能微孔板读板机 (Molecular Devices)

方法

激酶反应

步骤1.在1xIMAP反应缓冲液中加入DTT，并使DTT得终浓度达到1mM(1:100稀释100mM的DTT贮存液)，得到完整的反应缓冲液(CRB)；

步骤2.在CRB中制备400nM(4x)的FAM或TAMRA标记的多肽底物(1:50稀释原20uM的多肽贮存液)；

步骤3.在CRB中制备20uM(4x)的ATP(1:2500稀释原50mM的ATP贮存液)；

步骤4.准备一份终浓度为4x的酶梯度稀释液。对于激酶抑制剂试验，使用恒定浓度的酶，并制备出一组十字孢碱或其它激酶抑制剂的梯度稀释液。

步骤5.将下列试剂平行的加入4个复孔的激酶反应体系中：

• 5ul CRB或十字孢碱
• 5ul酶(对于没有添加酶的背景孔， 加入5ul CRB作为替代)
• 5ul 20uM的ATP贮存液
• 5ul 400 nM的多肽贮存液

步骤6.室温孵育1到1.5小时。

Note：如需获得更为详尽的说明，请参考 IMAP product insert 1.另外，对于多种激酶 的试验数据也可以登录以下网址进行查寻 http://www.moleculardevices.com/assayar chive/

校准标准品的制备

步骤1.在CRB中制备一份100nM的多肽贮存液(使用与激酶试验中相同的底物)；

步骤2.在CRB中制备一份100nM的磷酸多肽贮存液(使用与激酶试验中相同的磷酸化底物)；

步骤3.如图表1所示将多肽和磷酸化多肽贮存液按照一定规律混合后，制备校正标准品。样品量足够4个复孔所需；

Note：更多校正曲线配制及使用信息，请 参考IMAP Application Note #4, Developing Calibration Curves for IMAP2。

步骤4.各移出20ul校正标准品到4个复孔 中，包括一组只有20ul CRB的空白背 景样本。

结合反应

步骤1.将75%的结合缓冲液A与25%的结合缓冲液B混合，制备结合缓冲液；

Step 2: Prepare Binding Solu步骤2.将结合试剂以1:600倍稀释成结合缓冲液，得到结合反应液；

步骤3.移出60ul结合反应液到每个检测孔和校正标准孔中(包括背景孔样本)

步骤4.室温避光孵育1小时；

Set up template in SoftMax Pro and read plate on the SpectraMax M5 microplate reader:

Note: IMAP FP protocols for use with FAM- and TAMRA-labeled substrates are available in SoftMax Pro 5 in the Binding Assays protocol folder.

Step 1: Open the SoftMax Pro IMAP FP protocol specific to the fluorophore used. FAM and TAMRA protocols are available; adjust wavelength settings if a fluorophore other than FAM or TAMRA is used. Settings for the SpectraMax M5 microplate reader are shown in Table 2.

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

步骤2.设置一个试验模板，选定背景样品本 孔、激酶样品本孔、校正标准品孔。 激酶试验样品和校正标准品可分到 SoftMax Pro软件模板上预设好的8个 样本组中。在计算最小偏振值（mP） 之前，首先将只含有缓冲液的质控品 分到相应的背景组中后，使用软件自 动扣除其背景荧光值。

步骤3.把微孔板放到读板机里后---确保紫色托架（适配器）放置在微孔板托架上---然后点击Read读板。

结果

SoftMax Pro软件中预置的IMAP 荧光偏振 模板会自动计算出水平偏振均值和垂直偏 振均值(mP值)，总的荧光强度，标准方差 和CV值。每一组试验样品的组群一栏中， 模板会自动扣除背景样品的荧光偏振值， 然后计算出最小偏振mP值。也可根据相应 的校正曲线计算出样品磷酸化比率。(See IMAP Application Note #4, “Developing calibration curves for IMAP”)

图表2显示了使用 FAM-p34cdc2多肽获得 的一条LCK激酶的稀释度曲线。当用 SpectraMax M5微孔板读板机检测时，得 出酶的浓度范围从0.04至3.5 units/ml，最 小偏振值为303，同时Z'因子的值为0.87。 图表3显示了使用Analyst HT多功能读微孔 板读板机检测相同孔时，荧光偏振值为 336，Z'因子的值为0.95。两台仪器得到的 EC50值均为0.5 units/ml。

Figure 2. IMAP FP kinase assay on SpectraMax M5 microplate reader. Lck kinase dilution curve with FAM-p34cdc2-derived peptide from 0.04 to 3.5 units/mL enzyme, read on SpectraMax M5 (4-parameter curve fit). Error bars here and on all subsequent graphs are standard deviations.

Figure 3. IMAP FP kinase assay on Analyst HT. Lck kinase dilution curve with FAM-p34cdc2-derived peptide from 0.04 to 3.5 units/mL enzyme, read on Analyst HT (4-parameter curve fit).

如果使用非磷酸化和磷酸化的多肽样品作 为质控分别做一条校正曲线，可以将荧光 偏振值以百分比形式显示，得出其磷酸化 程度。(See Figure 4.)首先建立一条新的曲 线(软件默认名称为Graph#1)，分别使用荧 光偏振值(mP值)相对磷酸化百分比(浓度) 作为你的质控样品组绘制一条曲线。曲线 默认名称为“Plot#1.”在校正标准品组栏 里，新建一列后输入下面的公式，即： InterpX(‘Plot#1@Graph#1@IMAP FP_FAM’,AvgbkgsubmP)。并用‘IMAP FP_FAM’作为试验名称。

Figure 4. IMAP with FAM-labeled peptide. Calibration standard curve with FAM-p34cdc2-derived peptide and phosphopeptide, read on SpectraMax M5 (quadratic curve fit).

为了确定使用红色荧光染料进行IMAP激酶 试验的效果，另一个LCK激酶稀释曲线结果 的获得来源于TAMRA-p34cdc2多肽。(See Figure 5.)使用SpectraMax M5微孔板读板 机检测结果，酶的浓度范围是从0.04到3.5 units/ml，荧光偏振值(mP值)为258，同 时Z'因子的值为0.95。在Figure 6中，相同 的试验使用Analyst HT微孔板读板机进行 检测后，荧光偏振值(mP值)结果为270， 同时Z'因子的值为0.93。两个读板机得到的 EC50值都为0.4 units/ml，与FAM标记的 底物获得结果相一致。同样也建立了一条 相关的校正曲线(See Figure 7.)，TAMRA和 FAM相近的结果也可以让用户利用红色荧 光染料给我们带来的优势，即能够尽可能 降低化合物产生的背景荧光。

Figure 5. Lck kinase assay with TAMRA-labeled peptide on SpectraMax M5 microplate reader. Lck kinase dilution curve with TAMRA-p34cdc2-derived peptide, read on SpectraMax M5 (4-parameter curve fit).

Figure 6. Lck kinase assay with TAMRA-labeled peptide on Analyst HT. Lck kinase dilution curve with TAMRA-p34cdc2-derived peptide, read on Analyst HT (4-parameter curve fit).

Figure 7. IMAP with TAMRA-labeled peptide. Calibration standard curve with TAMRA-p34cdc2- derived peptide and phosphopeptide, read on SpectraMax M5 (linear curve fit).

Figure 8. Akt/PBKa kinase assay with FAM-labeled peptide on SpectraMax M5 microplate reader. Akt1/PKBα kinase dilution curve with FAM-Crosstide substrate, read on SpectraMax M5 (4-parameter curve fit).

Figure 9. Kinase inhibition on SpectraMax M5 microplate reader. Akt1/PKBα kinase inhibition curve with staurosporine and FAM-Crosstide substrate, read on SpectraMax M5. A five-parameter curve fit, available in SoftMax Pro 5, was used here.

总结

随着研究人员努力去探究激酶在细胞信号 转导通路和各种疾病中扮演的角色，以及 在许多疾病治疗中发挥的调节机制，他们 越来越希望获得非放射性的、均相的检测 方法，可用于试验优化和高通量药物的筛 选。荧光偏振试验可提供一种均相的、高 通量的方式来鉴定各种激酶的激活剂、抑 制剂和底物。使用SpectraMax M5多功能 微孔板读板机进行IMAP荧光偏振检测：获 得的结果精确性高、重复好、Z'因子值 高，双光栅型单色器系统方便使用者选择 不同荧光染料标记的多肽底物进行试验优 化，而无需额外的在选购滤光片。此外， 无论是用绿色或者红色荧光染料标记的底 物进行IMAP荧光偏振激酶试验，获得的结 果都与使用滤光片型单色器的微孔板读板 机上的检测值相一致。最后，SoftMax Pro 软件上预设的IMAP 荧光偏振模板为荧光偏 振数据的采集和分析提供了一种更为方便 的方式，并且这些模板也可用于不同荧光 染料分子的检测。

References

1. IMAP Akt Assay Kit Product Insert (Molecular Devices product #R8058).
2. Zhang, J.H., Chung, T.D.Y., and Oldenburg, K.R. (1999). A simple statistical parameter for use in evaluation and validation of highthroughput screening assays. J Biomol Screen 4(2): 67-73.

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