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FRET-based Stoichiometry Measurements of Protein Complexes in vitro

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Mar 2017



For a complete understanding of biochemical reactions, information on complex stoichiometry is essential. However, measuring stoichiometry is experimentally challenging. Our lab has developed a FRET-based assay to study protein complex stoichiometry in vitro. This assay, also known as Job plot, is set up as a continuous variation of the molar ratio between the two species, kept at constant total concentration. The FRET (Fluorescence Resonance Energy Transfer) between the two fluorescently-labeled proteins is measured and the stoichiometry is inferred from the sample with highest FRET signal. This approach allows us to assess complex stoichiometry in solution.

Keywords: Stoichiometry (化学计量学), FRET (FRET), Histones (组蛋白), Fluorescence (荧光), Complex formation (复合物形成), Job plot (工作曲线法)


Each biochemical reaction requires the interaction between two or more cellular components. The stoichiometry of these interactions is an important factor that regulates biochemical reactions in the cell. Experimental determination of complex stoichiometry is therefore critical to fully understand the biochemical and biophysical processes at work within cells.
Measuring stoichiometry has been experimentally challenging. For the interaction between large particles that lead to dramatic molecular weight changes, stoichiometry can be inferred by low-resolution structural analysis. These approaches include size-exclusion chromatography, multi-angle light scattering, analytical ultracentrifugation, which are techniques capable of providing accurate molecular weights of the particles. However, these methods require a considerable amount of material and are prone to error when small molecular weight changes are to be observed.

We have optimized an assay to measure complex stoichiometry in solution based on FRET (Fluorescence Resonance Energy Transfer). This assay, also known as Job plot (Huang, 1982) can be carried out with considerably less material and it is suitable for studying any complex formation, independent on the size of the two components. In this assay, samples are kept at constant total protein concentration, but with continuous variation of the molar ratio between the two components (Figure 1). The sample with the functional stoichiometry of the complex will display the highest FRET signal.

This is a powerful method that enables measurements of complex stoichiometry in solution and among components of any size. Because FRET measurements require both components of the complex to be fluorescently-labeled, a variety of controls are required to exclude potential artifact of the labeling procedure. Ideally single site labeling is advisable in this assay (D’Arcy et al., 2013; Mattiroli et al., 2017), but this is not always possible, as the protein structure may not be known for the components. We suggest performing functional assays with the labeled protein to confirm that the fluorophores do not alter its properties.

Materials and Reagents

  1. Low retention pipette tips (USA Scientific)
  2. PCR tubes (USA Scientific, catalog number: 1402-4308 )
  3. 384-well assay plate (Corning, catalog number: 3575 )
  4. PD-10 column (GE Healthcare, catalog number: 52130800 )
  5. Amicon Ultra centrifugal filter units, Ultra-15, MWCO 10 kDa (Merck, catalog number: UFC901008 )
  6. Unlabeled refolded H3-H4 (procedures explained in Dyer et al., 2004)
  7. Histone-binding protein (defined as Protein1 in this protocol)
  8. Alexa488-labeled refolded H3-H4 (procedures explained in Muthurajan et al., 2016) (Protein2)
  9. Atto647N maleimide (Sigma-Aldrich, catalog number: 05316-1MG-F )
    Note: Store at -20 °C in DMSO at 10 mM concentration.
  10. Dithiothreitol (DTT) (Gold Biotechnology, catalog number: DTT50 )
  11. Tris pH 7.5 (Fisher Scientific, catalog number: BP152-5 )
  12. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271-10 )
  13. EDTA (Fisher Scientific, catalog number: BP120-1 )
  14. TCEP (Gold Biotechnology, catalog number: TCEP10 )
  15. Nonidet P-40 (NP-40) (Sigma-Aldrich, catalog number: 74385 )
  16. CHAPS (Sigma-Aldrich, catalog number: C3023 )
  17. 1 M dithiothreitol (DTT; see Recipes)
  18. PD-10 buffer (see Recipes)
  19. Binding buffer (see Recipes)


  1. Pipettes (Gilson)
  2. Plate reader (BMG LABTECH, model: CLARIOstar )
  3. Centrifuge with plate adapter (Beckman Coulter, model: Allegra® X-22R , rotor: Beckman Coulter, model: S2096 )


  1. Excel Software


  1. Fluorescence labeling of the histone-binding protein
    1. In a minimum reaction volume of 400 µl, mix 10 µM of histone-binding protein with 10 µM of Atto647N maleimide dye, in PD-10 buffer. Rotate gently at 4 °C for 1 h. Protect the reaction from light.
    2. Quench the reaction with a final concentration of 10 mM DTT (see Recipes).
    3. Dilute the sample to 2 ml and apply it to a PD-10 column. Elute with 3.5 ml of PD-10 buffer (see Recipes). This step removes unconjugated dye from the solution.
    4. Concentrate the labeled protein to ~20 µM using Amicon Ultra (spinning at 3,500 x g at 4 °C) and store at 4 °C. Use within 2 days and freshly label a new aliquot for subsequent repeats of the assay.
    5. Protein function should always be validated after labeling with fluorescence dyes. This includes confirming that the labeled protein still binds to its partner with the same affinity.

  2. Assay design
    1. Reactions are set up in duplicate
    2. Each well contains a 40 µl reaction.
    3. Always include unlabeled controls as specified in Figure 1.

      Figure 1. Experimental setup. See also Supplemental file 1.

    4. Supplementary file 1 contains the worksheet for sample preparation.
    5. The total protein concentration in each well should remain constant, and should be at least 10 times higher than the binding constant (Kd) of the interaction between the two components. The relative molar ratio between the two components varies continuously.
      In the example in Supplementary file 1: the Kd is ~1 nM, hence we keep the total protein concentration in each tube at 150 nM.
      This means that in column 2, we have 150 nM Protein1 (i.e., Histone-binding protein), and in column 14 we have 150 nM Protein2 (i.e., H3-H4), while in column 8 we have 75 nM Protein1 (i.e., Histone-binding protein) and 75 nM Protein2 (i.e., H3-H4).

  3. Assay preparation
    1. Prepare 1 µM stock of each protein, labeled and unlabeled.
    2. Use the worksheet attached to prepare the stock solutions. These are prepared at twice the protein concentration required in the well reactions.
    3. Final well reactions are prepared by mixing 20 µl of Protein1 solution with 20 µl of Protein2 solution, leading to the desired final protein concentration, as they are diluted two-fold in this step.
    4. Mix well by pipetting, but avoid forming bubbles.
    5. Spin down the plate at 100 x g (RCF: relative centrifugal force) for 1 min.

  4. Data collection
    The fluorescence intensity is measured in a CLARIOstar machine (BMG labtech) using the following settings:
    1. Measure acceptor fluorescence:
      Excitation wavelength-bandwidth: 625-30 nm;
      Emission wavelength-bandwidth: 680-30 nm;
      Dichroic: automatic;
      Gain settings for this channel are adjusted automatically using the sample in wells B2, with highest acceptor dye concentration and no donor dye present.
    2. Measure FRET:
      Excitation wavelength-bandwidth: 488-15 nm;
      Emission wavelength-bandwidth: 680-30 nm;
      Dichroic: automatic;
      Gain settings for this channel are not adjusted at first, when an average value is used to gauge the region of highest FRET. Further measurements are repeated at different gain settings to confirm the sample with highest FRET and then this sample (in our example well C8) is used for the final automatic adjustment and measurement.
    3. Measure donor fluorescence:
      Excitation wavelength-bandwidth: 488-15 nm;
      Emission wavelength-bandwidth: 535-30 nm;
      Dichroic: automatic;
      Gain settings for this channel are adjusted automatically using the sample in wells A14, with highest donor dye concentration and no acceptor dye present.
      The plate-reader software will output the raw data in Excel format.

Data analysis

Data analysis to calculate the corrected FRET signal is performed as described in Hieb et al. (2012) and Winkler et al. (2012). We attached an example analysis file (Supplemental file 2). In summary:

  1. Perform buffer subtraction, as done in R1-AD29 in the attached analysis file (Supplemental file 2). Subtract the average intensity of the Buffer only samples (column 1) from each control and experimental measurement.
  2. Calculate donor bleed-through intensity, as done in Q-AD35 in the attached analysis file. Calculate the ratio between the FRET intensity in the sample where only the donor dye is present (row A) and the donor fluorescence intensity in the same sample. Average the values obtained in the two replicates.
  3. Calculate acceptor direct excitation intensity, as done in Q-AD37 in the attached analysis file. Calculate the ratio between the FRET intensity in the sample where only the acceptor dye is present (row B) and the acceptor fluorescence intensity in the same sample. Average the values obtained in the two replicates.
  4. Calculate the corrected FRET, as done in Q-AD39 and Q-AD40 in the attached analysis file. This is equal to:
    Measured FRET in the samples containing both dyes (row C)–donor bleed-through *donor signal in the same sample–acceptor direct excitation *acceptor signal in the same sample.
  5. The average of the corrected FRET from the two replicates is plotted with the Standard deviation (Figure 2).
    The stoichiometry is indicated by the sample with the highest FRET signal.
    In this case, the sample with Protein1 molar ratio 0.5, meaning with a 1:1 ratio of Protein1 and Protein2 has the highest measured FRET, suggesting that the complex between these two proteins contains one equivalent of each protein.

    Figure 2. Example plot of corrected FRET intensity


  1. Previous knowledge of the binding affinity (Kd) of the two binding partners is required for the proper setup of the experiment and accurate interpretation of the results.
  2. Other validated FRET pairs can be used with the described experimental setup.


  1. 1 M dithiothreitol (DTT)
    Dissolve 0.15425 g of DTT powder into 1 ml of ddH2O
    Resuspend by vortexing and store at -20 °C for maximum 2 weeks
  2. PD-10 buffer
    20 mM Tris pH 7.5 (pH measured at room temperature)
    300 mM NaCl
    1 mM EDTA
    1 mM TCEP
  3. Binding buffer
    20 mM Tris pH 7.5 (pH measured at room temperature)
    300 mM NaCl
    0.01% NP-40
    0.01% CHAPS
    1 mM EDTA
    1 mM TCEP


This protocol was adapted from D’Arcy et al., 2013. F.M. is funded by EMBO (ALTF 1267-2013) and the Dutch Cancer Society (KWF 2014-6649). Research in the Luger lab is funded by the Howard Hughes Medical Institute and NIH (GM067777). The authors declare no conflicts of interest or competing interests.


  1. D’Arcy, S., Martin, K. W., Panchenko, T., Chen, X., Bergeron, S., Stargell, L. A., Black, B. E. and Luger, K. (2013). Chaperone Nap1 shields histone surfaces used in a nucleosome and can put H2A-H2B in an unconventional tetrameric form. Mol Cell 51(5): 662-677.
  2. Dyer, P. N., Edayathumangalam, R. S., White, C. L., Bao, Y., Chakravarthy, S., Muthurajan, U. M. and Luger, K. (2004). Reconstitution of nucleosome core particles from recombinant histones and DNA. Methods Enzymol 375: 23-44.
  3. Hieb, A. R., D’Arcy, S., Kramer, M. A., White, A. E. and Luger, K. (2012). Fluorescence strategies for high-throughput quantification of protein interactions. Nucleic Acids Res 40: e33.
  4. Huang, C. Y. (1982). Determination of binding stoichiometry by the continuous variation method: the Job plot. Methods Enzymol 87: 509-525.
  5. Mattiroli, F., Gu, Y., Yadav, T., Balsbaugh, J. L., Harris, M. R., Findlay, E. S., Liu, Y., Radebaugh, C. A., Stargell, L. A., Ahn, N. G., Whitehouse, I. and Luger, K. (2017). DNA-mediated association of two histone-bound complexes of yeast Chromatin Assembly Factor-1 (CAF-1) drives tetrasome assembly in the wake of DNA replication. Elife 6.
  6. Muthurajan, U., Mattiroli, F., Bergeron, S., Zhou, K., Gu, Y., Chakravarthy, S., Dyer, P., Irving, T. and Luger, K. (2016). In vitro chromatin assembly: Strategies and quality control. Methods Enzymol 573: 3-41.
  7. Winkler, D.D., Luger, K. and Hieb, A. R. (2012). Quantifying chromatin-associated interactions: the HI-FI system. Methods Enzymol 512: 243-274.


为了全面了解生化反应,复杂的化学计量学信息是必不可少的。 然而,测量化学计量学在实验上是具有挑战性的。 我们的实验室已经开发了基于FRET的测定法来研究蛋白质复合体化学计量体外。 该测定法也被称为作业区,其被设定为两种物质之间的摩尔比的连续变化,保持恒定的总浓度。 测量两种荧光标记蛋白之间的FRET(荧光共振能量转移),并从具有最高FRET信号的样品中推断化学计量。 这种方法使我们能够评估溶液中复杂的化学计量。



这是获得溶液中复杂化学计量和任何大小组分之间测量的有力方法。因为FRET测量需要复合物的两个组分都被荧光标记,所以需要各种对照来排除标记过程的潜在伪影。理想的情况是单个位点的标记在该测定中是可取的(D'Arcy等人,2013; Mattiroli等人,2017),但这并不总是可能的,因为蛋白质结构可能不是已知的组分。我们建议使用标记的蛋白质进行功能测定,以确认荧光团不会改变其性质。

关键字:化学计量学, FRET, 组蛋白, 荧光, 复合物形成, 工作曲线法


  1. 低保留枪头(美国科学)
  2. PCR管(USA Scientific,目录号:1402-4308)。
  3. 384孔测定板(Corning,目录号:3575)。
  4. PD-10柱(GE Healthcare,目录号:52130800)
  5. Amicon超离心过滤装置,Ultra-15,MWCO 10 kDa(Merck,目录号:UFC901008)。
  6. 未标记的重折叠的H3-H4(在Dyer等人,2004中解释的程序)。

  7. 组蛋白结合蛋白(在此协议中定义为蛋白1)
  8. Alexa488标记的重折叠的H3-H4(在Muthurajan等人2016中解释的程序)(Protein2)。
  9. Atto647N马来酰亚胺(Sigma-Aldrich,目录号:05316-1MG-F) 等级:在-20℃下以10mM浓度储存在DMSO中。
  10. 二硫苏糖醇(DTT)(Gold Biotechnology,目录号:DTT50)。
  11. Tris pH 7.5(Fisher Scientific,目录号:BP152-5)。
  12. 氯化钠(NaCl)(Fisher Scientific,目录号:S271-10)。
  13. EDTA(Fisher Scientific,目录号:BP120-1)。
  14. TCEP(黄金生物技术,目录号:TCEP10)
  15. Nonidet P-40(NP-40)(Sigma-Aldrich,目录号:74385)
  16. CHAPS(Sigma-Aldrich,目录号:C3023)
  17. 1μM二硫苏糖醇(DTT;见食谱)。
  18. PD-10缓冲液(见食谱)
  19. 绑定缓冲区(请参阅食谱)


  1. 移液器(吉尔森)
  2. 读板器(BMG LABTECH,型号:CLARIOstar)
  3. 用Beckman Coulter,型号:Allegra X-22R,转子:Beckman Coulter,型号:S2096)离心。


  1. Excel软件


  1. 荧光标记组蛋白结合蛋白
    1. 在400μl的最小反应体积中,将10μM的组蛋白结合蛋白与10μM的Atto647N马来酰亚胺染料在PD-10缓冲液中混合。在4℃下轻轻转动1小时。保护光线下的反应。
    2. 用终浓度为10mM的DTT终止反应(参见食谱)。
    3. 将样品稀释至2 ml,并将其应用于PD-10色谱柱。用3.5ml的PD-10缓冲液洗脱(见食谱)。这一步从溶液中去除未结合的染料。
    4. 使用Amicon Ultra(在4℃以3.500×g旋转)将标记的蛋白质浓缩至约20μM,并在4℃下储存在2天内使用,并为随后的测定重复新鲜标记新的等分试样。
    5. 用荧光染料标记后,应始终对蛋白质功能进行验证。这包括标记的蛋白质仍然以相同的亲和力与其伴侣结合。

  2. 分析设计
    1. 反应设置重复。
    2. 每孔含有40μl的反应。
    3. 总是包含图1中指定的未标记的控件。

      图1.实验设置 。另请参阅补充文件1

    4. 补充文件1包含样本的代码表准备。
    5. 每个孔中的总蛋白质浓度应该保持不变,并且应该至少比两个组分之间的相互作用的结合常数(Kd)高10倍。
      两种成分的相对摩尔比连续变化 在
      补充文件1的代码: Kd是〜1 nM,所以我们将每个试管中的总蛋白质浓度保持在150 nM。
      此bedeutet,DASS 2栏,我们有150 nM的蛋白1(即,组蛋白结合蛋白),并在第14栏,我们有150 nM的蛋白2(即,H3-H4 ),而在第8栏,我们有75 nM的蛋白1(即,组蛋白结合蛋白)和75 nM的蛋白2(即,H3-H4)。

  3. 测定准备
    1. 准备1μM的每种蛋白质,标记和未标记。
    2. 使用附带的工作表来准备库存解决方案。
    3. 20μl含有20μl蛋白质2溶液的蛋白质1溶液,导致所需的最终蛋白质浓度,在此步骤中稀释两倍。

    4. 用移液器充分混合,但避免形成气泡
    5. 以100×g(RCF:相对离心力)旋转板1分钟。

  4. 数据收集
    使用以下设置在CLARIOstar机器(BMG labtech)中测量荧光强度:
    1. 测量受体荧光:
      激发波长带宽:625-30 nm;
      发射波长带宽:680-30 nm;
    2. 测量FRET:
      激发波长带宽:488-15 nm;
      发射波长带宽:680-30 nm;
    3. 测量供体荧光:
      激发波长带宽:488-15 nm;
      发射波长带宽:535-30 nm;


FRET信号如在(2012)和Winkler(et al。(2012))中所述进行。我们进行固定的例子分析文件(补充文件2 )。总结:

  1. 执行缓冲减法,在R1-AD29中附加的文件分析进行(补充文件2 )。从每个对照和实验测量中减去仅缓冲液样品(第1列)的平均强度。
  2. 计算供体渗透强度,如附件分析文件中的Q-AD35所示。计算仅存在供体染料(A行)的样品中的FRET强度与同一样品中的供体荧光强度之间的比率。平均两次重复获得的值。
  3. 计算受主直接激发强度,如附件分析文件中的Q-AD37所示。计算仅存在受体染料(B行)的样品中的FRET强度与同一样品中的受体荧光强度之间的比率。平均两次重复获得的值。
  4. 计算校正后的FRET,如附件分析文件中的Q-AD39和Q-AD40所示。这等于:
    在同一样品中同一样品 - 受体直接激发*受体信号中同时含有两种染料(C行)的样品中发出信号FRET-出血渗透*供体信号。

  5. 来自两个重复的FRET用标准偏差绘制(图2)。



  1. 先前对两种结合伴侣的结合亲和力(Kd)的知识是正确设定实验和结果的准确解释所必需的。
  2. 其他经过验证的FRET对可以与描述的实验设置一起使用。


  1. 1 M二硫苏糖醇(DTT)。
    将0.15425克DTT粉末溶解于1毫升ddH2O中 涡旋振荡,-20°C保存2周。
  2. PD-10缓冲区
    20mM Tris pH 7.5(在室温下测量的pH)。
    1mM EDTA。
    1mM TCEP。
  3. 绑定缓冲区
    20mM Tris pH 7.5(在室温下测量的pH)。
    1mM EDTA。
    1mM TCEP。


该协议是从D'Arcy等人,2013年改编的。由EMBO(ALTF 1267年至2013年)和荷兰癌症协会(KWF 2014-6649)资助。 Luger实验室的研究由Howard Hughes医学研究所和NIH(GM067777)资助。作者声明不存在利益冲突或利益冲突。


  1. 达西,S.,马丁,K. W.,Panchenko,T.,陈,X.,伯杰龙,S.,Stargell,洛杉矶,黑色,B. E.和卢杰,K.(2013)。 伴侣Nap1时遮蔽在核小体组蛋白使用表面和可以把H2A-H2B非常规四聚体形式。 51(5):662-677。
  2. 代尔,P. N.,Edayathumangalam,R.S。,白色,C. L.,宝,Y.,Chakravarthy,S.,Muthurajan,U. M.和卢格,K。(2004)。从重组组蛋白和DNA。 酶学方法<核小体核心颗粒的重建/ 375:23-44。
  3. 斩,A. R.,达西,S.,克莱默,M. A.,白色,A. E.和卢格,K。(2012)。 核酸研究target="_blank">荧光策略 40:e33。
  4. Huang,C.Y.(1982)。 测定:作业积代码。方法Enzymol 87:509-525。
  5. Mattiroli,F.,顾,Y.,亚达夫T.,Balsbaugh,JL,哈里斯,MR,芬德利,ES,刘,Y.,Radebaugh,CA,Stargell,LA,安贞焕,NG,白宫和一鲁格,K.(2017)。的酵母染色质装配因子1(CAF-1两组蛋白结合的复合物的 DNA介导的缔合)在DNA复制后驱动tetrasome组装。 6.
  6. Muthurajan,U.,Mattiroli,F.伯杰龙,S.,周,K.,谷,Y.,Chakravarthy,S.,代尔,P.,欧文,T。和卢格,K。(2016)。 体外染色质装配。策略和质量控制 Methods Enzymol 573:3-41。
  7. Winkler,D.D.,Luger,K。和Hieb,A.R。(2012)。 量化染色质相关的相互作用:对HI-FI系统的代码酶学方法。 512:243-274。
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Copyright Mattiroli et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Mattiroli, F., Gu, Y. and Luger, K. (2018). FRET-based Stoichiometry Measurements of Protein Complexes in vitro. Bio-protocol 8(3): e2713. DOI: 10.21769/BioProtoc.2713.
  2. Mattiroli, F., Gu, Y., Yadav, T., Balsbaugh, J. L., Harris, M. R., Findlay, E. S., Liu, Y., Radebaugh, C. A., Stargell, L. A., Ahn, N. G., Whitehouse, I. and Luger, K. (2017). DNA-mediated association of two histone-bound complexes of yeast Chromatin Assembly Factor-1 (CAF-1) drives tetrasome assembly in the wake of DNA replication. Elife 6.