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Determination of Protein-DNA (ZMYND11-DNA) Interaction by a Label-Free Biolayer Interferometry Assay
采用无标记生物层干涉量度分析法测定蛋白质-DNA(ZMYND11-DNA)的相互作用   

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Abstract

This protocol describes a robust technique for the measurement of ZMYND11-DNA interaction by a label-free Biolayer Interferometry (BLI). ZMYND11 is a novel histone reader protein that specifically recognizes H3.3K36me3 via its tandem Bromodomain, zinc-finger and PWWP domain (BP). ZMYND11 links the histone-variant-mediated transcription elongation control to tumour suppression and may therefore represent a novel class of drug targets. Like other PWWP domains, ZMYND11 PWWP domain shows highly positively charged surface and interacts with DNA. Previously reported methods include NMR, FP or EMSA. Biolayer interferometry (BLI) is an emerging technology for analyzing all kinds of biomolecular interactions, such as protein-protein and protein-DNA binding. BLI allows for the real time monitoring of the interactions between biomolecules without the need for reagents with enzymatic, fluorescent, or radioactive labels. The technology is based upon the changes in interference pattern of light reflected from the surface of an optical fiber when materials bind to the tip of the fiber. The technique represents an alternative to technologies such as surface plasmon resonance, providing a simple platform that enables label-free monitoring of biomolecular interactions without the use of flow cells. Label-free biosensor methods provide information on binding, kinetics, concentration, and the affinity of an interaction.

Keywords: Biolayer interferometry(BioLayer干涉), Protein-DNA interaction(蛋白质与DNA的相互作用), Label free(无标记), Epigenetic regulation(表观遗传调控), PWWP domain(重构)

Materials and Reagents

  1. Purified proteins of interest untagged or with tags such as GST, Flag, Biotin or His tag, depending on which kind of sensor to use (refer to manufacturer’s manual)
    Note: Here GST-tagged ZMYND11-BP protein is used (Wen et al., 2014).
  2. Two complementary single stranded DNA (Life Technologies, InvitrogenTM)
  3. MilliQ water
  4. 10 mM glycine (pH 1.0~2.0)
  5. BLI binding buffer (see Recipes)
  6. DNA solutions (see Recipes)

Equipment

  1. Anti-GST antibody-coated biosensor tray (Pall Corporation, catalog number: 18-5096 )
  2. 96-well black microtiter plate (Greiner Bio-One, catalog number: 655209 )
  3. 1.5 ml Eppendorf microfuge tubes (Axygen, catalog number: MCT150C )
  4. Octet RED96 (fortebio) (Pall Corporation, catalog number: 30-5048) (Wen et al., 2014)

Software

  1. Octet Data analysis 7.0

Procedure

  1. Getting started
    1. Turning on the system:
      Turn on the computer.
      Next, make sure the sensor and sample positions are clear inside the Octet and turn on the power supply.
      Finally, launch the Octet Data Acquisition software. Octet will initialize and home the optics box.
      Warming up the Octet.
      The Octet should be turned an hour prior to an experiment being run to allow for the lamp to warm up.
    2. Setting up experiment.
      Use 200 µl samples in each well.
      Pre-wet the sensors in BLI binding buffer for 30 min (time may vary depending on sensor – consult product insert):
      Place 200 µl BLI binding buffer in each of eight wells of a 96-well black microtiter plate Insert the biosensors tray onto the 96-well black microtiter plate (Figure 1).


      Figure 1. Flowchart to show how to pre-wet the BLT biosensors

  2. Assay
    1. Start by defining the number of conditions you need to test, including appropriate controls such as protein sample in buffer only, or DNA sample in buffer only. This will determine the total volume of protein and DNA needed to conduct the entire experiment. Each well is conducted in a total volume of 200 μl.
    2. Pre-wet the biosensors in BLI binding buffer for at least 30 min. Biolayer interferometry experiments were conducted using an Octet Red, manufactured by ForteBio (Figure 2). The instrument can measure the signal from eight sensors simultaneously.


      Figure 2. The ForteBio Octet Red96 system (Left), biosensor tray (Middle) and resulting signal curves shown (Right)

    3. Sample plate definition

      1
      2
      3
      4
      5
      6
      7
      8
      9
      10
      11
      12
      A
      AB
      GST-ZMYND11
      AB
      S1
      AB





      RE
      AB
      B
      AB
      GST-ZMYND11
      AB
      S2
      AB





      RE
      AB
      C
      AB
      GST-ZMYND11
      AB
      S3
      AB





      RE
      AB
      D
      AB
      GST-ZMYND11
      AB
      S4
      AB





      RE
      AB
      E
      AB
      GST-ZMYND11
      AB
      S5
      AB





      RE
      AB
      F
      AB
      GST-ZMYND11
      AB
      S6
      AB





      RE
      AB
      G
      AB
      GST-ZMYND11
      AB
      S7
      AB





      RE
      AB
      H
      AB
      GST-ZMYND11
      AB
      AB
      AB





      RE
      AB
      AB=Assay buffer, S=sample; S1, 2…7 are different concentrations of different DNA samples from low to high, RE=regeneration buffer
    4. Assign the sample type to the sample plate by left click the lane numbers. Here each lane of the plate is defined as ‘buffer (B), loading (L), buffer (B), sample (colored pink), buffer (B),…regeneration (R), buffer (B)’ (Figure 3).


      Figure 3. Sample plate definition

    5. Prepare 30 µg/ml GST-ZMYND11 proteins in BLI binding buffer in a total volume of 200 μl/well multiplied by the number of conditions you need to test.
    6. Prepare a dilution series (six here) of your DNA sample in binding buffer ranging from 3.125 to 100 μM final concentrations in a total volume of 500 μl in case you want duplicate.
    7. Dispense 200 μl of protein/buffer in each well. Prepare samples in duplicate.
    8. Sensor assignment. Choose the right positions where the sensors are assigned.
    9. Insert the Sensor Tray into the Sensor Tray Holder and the Sample Plate into the Sample Plate Holder (refer to Figure 4 and manufacturer’s manual).


      Figure 4. The internal structure of Octet Red96 system

      Notes:
      1. Sample & concentration settings are quite important. Typical final protein concentration is around 20-50 µg/ml (determined by UV280 absorbance). According to the binding affinity (KD) to test, it is recommended that DNA dilution series are set in such a concentration range that cover 0.5x KD ~ 20x KD (the DNA concentration is determined by UV260 absorbance) and pre-test to determine the sample concentration and KD are necessary. The system used here has 8 sensors and uses a 96-well sample plate. Thus, interaction between a sensor-immobilized biomolecule and its binding partner in solution can be tested under different conditions with multiple sensors in parallel. For example, interactions were measured with six different concentrations of the binding partner in parallel.
        Pre-test 1:
        1. Relative high concentration (10x KD/100x KD).
        2. Positive control.
        3. Blank control.
        4. Yes/No binding. Yes samples go next round detection.
        Pre-test 2:
        1. Yes samples go this round or KD test directly according to the researcher.
        2. Find the proper concentration range (using a large dilution factor such as 10 or 5
          to determine the highest and lowest concentration) if pre-test 2 performed.
        KD test:
        1. No less than 4 concentrations should be included in sample dilution series with
          a dilution factor 2 or 3.
        2. Positive/negative control (not necessary).
        3. Blank control.
      2. Once the sensors were prepared, the assays were quite easy to conduct because the instrument can perform all of the assay functions without attention from the analyst. For example, the 96-well plate does not need to be removed and washed with a separate instrument at various stages in the analysis. Also, substrate does not need to be prepared and added, etc. These are potential advantages of the biolayer interferometry technique (refer to manufacturer’s manual).

  3. Program
    All experiments consisted of repeated cycles, each having six steps:
    1. Incubation with the BLI binding buffer to establish a baseline signal (baseline, 2 min).
    2. Incubation with protein sample with putative tags to be immobilized onto the biosensors (loading, 5 min).
    3. Incubation with the BLI binding buffer again to remove unbound proteins to a baseline signal (baseline, 2 min).
    4. Incubation with the DNA sample (association, 5 min).
    5. Incubation with the BLI binding buffer to remove the DNA sample (dissociation, 5 min) followed by a 90 sec regeneration step using glycine.
      This is can be performed by assay definition. Here the assay includes the following steps: baseline-loading-baseline-association-dissociation-regeneration-neutralization.
      Connect each step to corresponding lane and review the assay before click Run button.
      Each assay cycle takes about 15 min. All experiments were conducted at 25 °C, and the total volume of the test solution in all cases was 0.2 ml. The samples are stirred by orbital shaking throughout the assay (1,000 rpm. vibration here).

  4. Data analysis
    1. Save data as response upon binding (nm) vs time. Export file in a format suitable for import into Microsoft Excel or Origin for analysis.
      Collected experimental data is analyzed according to the fitting algorism supplied by vendor of the Octet RED96 system. Usually, steady-state analysis shows great agreement with binding kinetics analysis and 1:1 fitting model is employed here.
      Note: Check the chi-square value and R
      2 value supplied by the software. Chi-square/DOF (degrees of freedom): if using 1:1 fitting model, the DOF=1, and chi-square value should be equal to 3.5. Thus, chi-square<3.5 and 1>R2>0.8 indicate a reasonable data.
    2. Below is an example of a typical BLI experiment.


      Figure 5. Experimental setup of BLI assay includes plate definition, assay definition and sensor assignment


      Figure 6. Runtime binding chart and data analysis of a typical BLI. GST-tagged BP protein (30 µg/ml) was immobilized onto anti-GST sensor and a series of dilution sample of methylated DNA (100 µM, 50 µM, 25 µM, 12.5 µM, 6.25 µM, 3.125 µM, from top to bottom) was used as the binding partner. Steady state analysis and 1:1 fitting model was employed here.

Notes

  1. The instrument can measure the response from eight sensors simultaneously. From preliminary experiments it was clear that substantial variability existed in the output from the individual sensors. The variability in response between sensors suggested that using certain sensors for generating the calibration curve and other sensors for measuring the responses from unknown (or spiked) samples would likely lead to poor quantitation of interaction.
  2. The optimization of BLI binding buffer aims to get your protein and the binding most stable. Theoretically, BLI is compatible with any kind of buffer component. Of noted is that if using biotinylated molecules, Tris should not be used. The optimal BLI binding has to be determined before BLI binding buffer.

Recipes

  1. BLI binding buffer
    The composition of the binding buffer used can be optimized according to the stability and activity of each protein of interest.
    The following recipe was used to study ZMYND11
    25 mM NaH2PO4 (pH 7.0)
    150 mM KCl
    2 mM MgCl2
  2. DNA solutions
    Two complementary single stranded DNA were chemically synthesized. Double stranded DNA was obtained by annealing following the protocol below:
    1. Mix equal volumes of both complementary oligos (at equimolar concentration of 100 mM) in a 1.5 ml microfuge tube.
    2. Place tube in a standard heatblock at 90-95 °C for 3-5 min.
    3. Remove the heat block from the apparatus and allow to cool to room temperature (or at least below 30 °C) on the workbench. Slow cooling to room temperature should take 45-60 min.
    4. Stored on ice or at 4 °C until ready to use.
      50 mM stock prepared in annealing buffer: 20 mM Tris (pH 7.0), 50 mM NaCl and 1 mM EDTA

Acknowledgments

We thank W. Chu at Tsinghua the China National Center for Protein Sciences Beijing for providing facility support and technical assistance. This work was supported by grants to X. S. (CPRIT RP110471, Welch G1719, American Cancer Society RSG-13-290-01-TBE, and National institutes of Health (NIH)/MDACC CCSG CA016672), H. L. (The Major State Basic Research Development Program in China, 2011CB965300 and Program for New Century Excellent Talents in University), W. L. (CPRIT RP110471, NIH R01HG007538), B. L. (NIH R01GM090077, Welch I1713), Y. L. (China Postdoctoral Science Foundation, 2012M510413) and H. W. (MD Anderson IRG, Center for Cancer Epigenetics pilot grant). Y. L. is a Tsinghua-Peking Center for Life Sciences postdoctoral fellow. W.L. is a recipient of a Duncan Scholar Award and X.S. is a recipient of a Kimmel Scholar Award.

References

  1. Abdiche, Y., Malashock, D., Pinkerton, A. and Pons, J. (2008). Determining kinetics and affinities of protein interactions using a parallel real-time label-free biosensor, the Octet. Anal Biochem 377(2): 209-217.
  2. Abdiche, Y., Malashock, D., Pinkerton, A. and Pons, J. (2009). Exploring blocking assays using Octet, ProteOn, and Biacore biosensors. Anal Biochem 386(2): 172-180.
  3. Do, T., Ho, F., Heidecker, B., Witte, K., Chang, L. and Lerner, L. (2008). A rapid method for determining dynamic binding capacity of resins for the purification of proteins. Protein Expr Purif 60(2): 147-150.

简介

该协议描述了用于通过无标记生物分子干涉测量法(BLI)测量ZMYND11-DNA相互作用的鲁棒技术。 ZMYND11是一种新型组蛋白阅读器蛋白,通过其串联溴结构域,锌指和PWWP结构域(BP)特异性识别H3.3K36me3。 ZMYND11将组蛋白变体介导的转录伸长控制与肿瘤抑制联系起来,因此可能代表一种新型的药物靶标。像其他PWWP域一样,ZMYND11 PWWP域显示高度带正电荷的表面,并与DNA相互作用。先前报道的方法包括NMR,FP或EMSA。生物层干涉法(BLI)是用于分析各种生物分子相互作用(例如蛋白质 - 蛋白质和蛋白质-DNA结合)的新兴技术。 BLI允许实时监测生物分子之间的相互作用,而不需要具有酶,荧光或放射性标记的试剂。该技术基于当材料结合到光纤的尖端时从光纤的表面反射的光的干涉图案的变化。该技术代表了诸如表面等离子体共振等技术的替代方法,提供了一种简单的平台,其能够在不使用流动池的情况下实现生物分子相互作用的无标记监测。无标记的生物传感器方法提供关于结合,动力学,浓度和相互作用的亲和力的信息。

关键字:BioLayer干涉, 蛋白质与DNA的相互作用, 无标记, 表观遗传调控, 重构

材料和试剂

  1. 根据使用的传感器类型(参见制造商手册),未标记的或具有标签(例如GST,Flag,Biotin或His标签)的感兴趣的纯化蛋白质。 注意:这里使用GST标记的ZMYND11-BP蛋白(Wen等,2014)。
  2. 两个互补的单链DNA(Life Technologies,Invitrogen TM
  3. MilliQ水
  4. 10mM甘氨酸(pH 1.0〜2.0)
  5. BLI绑定缓冲区(参见配方)
  6. DNA解决方案(参见配方)

设备

  1. 抗GST抗体包被的生物传感器托盘(Pall Corporation,目录号:18-5096)
  2. 96孔黑色微量滴定板(Greiner Bio-One,目录号:655209)
  3. 1.5ml Eppendorf微量离心管(Axygen,目录号:MCT150C)
  4. Octet RED96(fortebio)(Pall Corporation,目录号:30-5048)(Wen等人,2014)

软件

  1. Octet数据分析7.0

程序

  1. 开始使用
    1. 打开系统:
      打开电脑。
      接下来,确保传感器和样品位置在Octet内清晰,并打开电源。
      最后,启动Octet数据采集软件。八位字节将初始化和回家光学盒 预热八位字节。
      在运行实验之前,应该将Octet转动一个小时,以使灯泡预热。
    2. 设置实验。
      在每个孔中使用200μl样品 在BLI结合缓冲液中预湿润传感器30分钟(时间可能因传感器而异 - 咨询产品插页):
      在96孔的8个孔的每个孔中放置200μlBLI结合缓冲液 黑色微量滴定板将生物传感器托盘插入96孔黑色  微量滴定板(图1)

      图1.显示如何预润湿BLT生物传感器的流程图

  2. 测试
    1. 首先定义您需要测试的条件数,包括 适当的对照例如仅在缓冲液中的蛋白质样品或DNA 仅在缓冲液中。 这将确定蛋白质的总体积 和进行整个实验所需的DNA。 进行每个孔 总体积为200μl。
    2. 在BLI中预湿润生物传感器 结合缓冲液至少30分钟。 生物层干涉测量实验 使用由ForteBio制造的Octet Red(图2)进行。 的 仪器可同时测量八个传感器的信号。


      图2。 ForteBio Octet Red96系统(左),生物传感器托盘(中)和显示的结果信号曲线(右)

    3. 样品板定义

      1
      2
      3
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      6
      7
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      9
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      12
      A
      AB
      GST-ZMYND11
      AB
      S1
      AB





      RE
      AB
      B
      AB
      GST-ZMYND11
      AB
      S2
      AB





      RE
      AB
      C
      AB
      GST-ZMYND11
      AB
      S3
      AB





      RE
      AB
      D
      AB
      GST-ZMYND11
      AB
      S4
      AB





      RE
      AB
      E
      AB
      GST-ZMYND11
      AB
      S5
      AB





      RE
      AB
      F
      AB
      GST-ZMYND11
      AB
      S6
      AB





      RE
      AB
      G
      AB
      GST-ZMYND11
      AB
      S7
      AB





      RE
      AB
      H
      AB
      GST-ZMYND11
      AB
      AB
      AB





      RE
      AB
      AB =测定缓冲液,S =样品; S1,2 ... 7是不同的浓度 不同的DNA样品从低到高,RE =再生缓冲液
    4. 通过左键单击泳道将样品类型分配到样品板 数字。 这里,板的每个通道被定义为"缓冲器(B),装载 (L),缓冲液(B),样品(粉红色),缓冲液(B),再生 缓冲液(B)'(图3)。


      图3。 样品板定义

    5. 制备30μg/ml GST-ZMYND11 蛋白质在BLI结合缓冲液中,总体积为200μl/孔 乘以您需要测试的条件数。
    6. 准备   您的DNA样品在结合缓冲液中的稀释系列(此处为6个) 终浓度范围为3.125至100μM,总体积为 500μl,如果你想要重复。
    7. 在每个孔中分配200μl的蛋白质/缓冲液。 一式两份制备样品。
    8. 传感器分配。 选择分配传感器的正确位置。
    9. 将传感器托盘插入传感器托盘支架和样品 将板放入样品板支架(参见图4和制造商手册)。


      图4。 Octet Red96系统的内部结构

      注意:
      1. 示例&浓度设置相当重要。典型 最终蛋白质浓度为约20-50μg/ml(通过UV280测定 吸光度)。根据测试的结合亲和力(KD),它是 推荐将DNA稀释系列设定为这样的浓度 范围,其覆盖0.5x KD〜20x KD(确定DNA浓度 通过UV260吸光度)和预测试以确定样品浓度 和KD是必要的。这里使用的系统有8个传感器,并使用a 96孔样品板。因此,传感器固定化之间的相互作用 生物分子及其在溶液中的结合配偶体可以在下进行测试 不同条件下并联多个传感器。例如, 相互作用用六种不同浓度的 绑定合作伙伴。
        预测试1:
        1. 相对高浓度(10×KD/100×KD)。
        2. 积极控制。
        3. 空白控制。
        4. 是/无约束。 是样品进行下一轮检测。
        预测试2:
        1. 根据研究者的情况,直接进行本轮或KD测试。
        2. 找到适当的浓度范围(使用大的稀释因子,如10或5
          以确定最高和最低浓度)如果进行了预测试2
        KD测试:

        1. 的样品稀释系列中应包含不少于4个浓度
        2. 正/负控制(不必要)。
        3. 空白控制
      2. 一旦准备好传感器,测定就相当容易 因为仪器可以执行所有的测定功能 没有分析师的注意。 例如,96孔板 不需要移除并用各种仪器进行清洗 阶段的分析。 此外,基底不需要制备和 添加等。这些是生物层的潜在优点 干涉测量技术(请参阅制造商手册)。

  3. 程序
    所有实验由重复循环组成,每个循环有六个步骤:
    1. 与BLI结合缓冲液孵育以建立基线信号(基线,2分钟)
    2. 用蛋白质样品与假定的标签孵育以固定在生物传感器上(加载,5分钟)
    3. 再次用BLI结合缓冲液温育以除去未结合的蛋白质至基线信号(基线,2分钟)
    4. 与DNA样品孵育(缔合,5分钟)
    5. 用BLI结合缓冲液温育以除去DNA样品 (解离,5分钟),然后使用90秒再生步骤 甘氨酸。
      这可以通过测定定义来进行。 这里 测定法包括以下步骤: 基线加载 - 基线 - 结合 - 解离 - 再生 - 中和 将每个步骤连接到相应的泳道,然后单击运行按钮之前查看测定 每个测定循环大约需要15分钟。 所有实验均在 25℃,所有情况下的试验溶液的总体积为0.2 ml。 在整个测定中通过轨道摇动搅拌样品 (这里为1000rpm)。

  4. 数据分析
    1. 保存数据作为响应绑定(nm)对时间。 以格式导出文件   适合导入到Microsoft Excel或Origin进行分析 根据拟合分析收集的实验数据 由Octet RED96系统的供应商提供的算法。 通常, 稳态分析显示与绑定动力学非常一致 分析和1:1拟合模型。
      注意:检查 卡方值和由软件提供的R 2 值。 卡方/DOF (自由度):如果使用1:1拟合模型,DOF = 1和 卡方值应等于3.5。 因此,卡方< 3.5和 1> R2> 0.8表示合理的数据。
    2. 以下是典型的BLI实验的示例。


      图5. BLI测定的实验设置包括平板定义,测定定义和传感器分配


      图6.运行时绑定图表和典型BLI的数据分析。将GST标记的BP蛋白(30μg/ml)固定在抗GST传感器 和一系列甲基化DNA的稀释样品(100μM,50μM,25μM,   12.5μM,6.25μM,3.125μM,从上到下)用作结合   伙伴。 这里采用稳态分析和1:1拟合模型。

笔记

  1. 仪器可以同时测量八个传感器的响应。从初步实验可以清楚地看出,来自各个传感器的输出存在实质性变化。传感器之间响应的变化性表明使用某些传感器产生校准曲线和其他传感器用于测量未知(或加标)样品的响应可能导致较差的相互作用定量。
  2. BLI结合缓冲液的优化目的是使您的蛋白质和结合最稳定。理论上,BLI与任何种类的缓冲器组件兼容。值得注意的是,如果使用生物素化分子,不应使用Tris。最佳BLI结合必须在BLI结合缓冲液之前测定

食谱

  1. BLI绑定缓冲区
    所使用的结合缓冲液的组成可以根据每种感兴趣的蛋白质的稳定性和活性来优化 以下食谱用于研究ZMYND11
    25mM NaH 2 PO 4(pH 7.0) 150 mM KCl
    2mM MgCl 2/
  2. DNA溶液
    化学合成两条互补的单链DNA。 按照以下方案通过退火获得双链DNA:
    1. 在1.5ml微量离心管中混合等体积的两种互补寡核苷酸(等摩尔浓度为100mM)。
    2. 将管在标准加热块中在90-95℃下3-5分钟。
    3. 从设备中取出加热块,让其冷却到室温 温度(或至少低于30℃)。 缓慢冷却至 室温需要45-60分钟。
    4. 储存在冰上或4℃,直到准备使用。
      在退火缓冲液中制备的50mM储液:20mM Tris(pH 7.0),50mM NaCl和1mM EDTA

致谢

我们感谢清华大学中国北京蛋白质科学中心提供设施支持和技术援助。这项工作得到了XS(CPRIT RP110471,Welch G1719,美国癌症协会RSG-13-290-01-TBE和国家卫生研究院(NIH)/MDACC CCSG CA016672),HL(主要国家基础研究发展(中国博士后科学基金,2012M510413)和HW(MD安德森IRG,中国博士后科学基金,2012M510413)和WL(CPRIT RP110471,NIH R01HG007538),BL(NIH R01GM090077,Welch I1713)癌症中心表观遗传学试点授予)。 Y.L.是清华北京生命科学博士后研究中心。 W.L.是邓肯学者奖的接受者。是Kimmel学者奖的获得者。

参考文献

  1. Abdiche,Y.,Malashock,D.,Pinkerton,A。和Pons,J。(2008)。 使用并行实时无标记生物传感器Octet确定蛋白质相互作用的动力学和亲和力。 Anal Biochem 377(2):209-217。
  2. Abdiche,Y.,Malashock,D.,Pinkerton,A。和Pons,J。(2009)。 使用Octet,ProteOn和Biacore生物传感器探索阻断测定法 Anal Biochem 386(2):172-180。
  3. Do,T.,Ho,F.,Heidecker,B.,Witte,K.,Chang,L.and Lerner,L。(2008)。 确定用于纯化蛋白质的树脂的动态结合能力的快速方法。 em> Protein Expr Purif 60(2):147-150。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Li, Y., Wen, H., Shi, X. and Li, H. (2015). Determination of Protein-DNA (ZMYND11-DNA) Interaction by a Label-Free Biolayer Interferometry Assay. Bio-protocol 5(4): e1402. DOI: 10.21769/BioProtoc.1402.
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