Fortebio生物分子相互作用检测平台-中文版
Octet BLI大分子结合动力学检测实验设计-Non-Small Molecule Kinetics
Kinetics Assay:概述：Octet系统可检测生物分子间相互作用，其中RED系列检测分子量下限可达150Da，而QK系列则可检测5kDa以上分子。
检测样品为分子量150 Da~1000 Da的小分子以及小于5kDa的分子，采用RED系列实现；分子量大于5kDa时，RED系列及QK系列均可满足要求。
该SOP默认待测样品为大分子，为其动力学检测提供通用标准化流程，针对具体分子及特殊情况，可能需要进一步考虑更多细节及优化。
考量及建议：1.采用Octet系统检测大分子时，应当根据样品特性综合考虑，以选择适当sensor（例如，SA sensor可loading生物素化的蛋白、抗体、化合物、核酸等以检测与之相互作用的大分子样品，而AHC sensor可直接loading人抗后检测与之相互作用的分子）；所使用sensor批次相同。
如用户对于sensor选择有任何疑问，请随时与ForteBio应用科学家联系。
2.蛋白或抗体生物素化操作请参阅ForteBio技术说明书。
化合物及核酸、糖类分子生物素化一般需合成实现。
核酸及糖类分子生物素化也可依据Pierce试剂盒操作实现。
3.Assay buffer没有特定要求，便于样品的稳定保存即可；值得注意的是，由于ForteBio大多通过氨基生物素化实现欧联，因此需生物素化样品的buffer不可为含Tris等氨基的缓冲液。
客户可根据需要采用Pierce相关试剂盒实现羧基端生物素化。
4.设置protocol时，一般需要1根sensor loading后在检测buffer中检测基线，作为空白对照。
5.用于loding的生物素化蛋白浓度可采用20ug-50ug/ml。
6.建议对样品进行两轮筛选。
例如，第1轮宽泛筛选，浓度为100,10,1,0.1uM，确定样品大概Kd范围；第2轮更精细筛选：30,10,3.3,1.1uM，获取高度可信的Kd。
（注意：所用浓度仅用于发挥导向性作用，实际浓度取决于初筛所获得Kd）。
fortebio
Fortebio技术概览引言Fortebio是一家生物科技公司，致力于提供先进的生物分析和生物传感技术解决方案。
该公司的核心技术包括光学生物传感技术，用于监测生物分子的相互作用。
本文将对Fortebio 公司的技术进行概览，并介绍其应用领域和优势。
技术原理Fortebio的核心技术基于表面等离子体共振（Surface Plasmon Resonance，SPR）现象，这是一种用于研究生物分子相互作用的光学技术。
SPR技术通过检测生物分子的表面等离子体共振现象，可以实时监测和分析生物分子之间的相互作用。
Fortebio的SPR技术基于Biolayer Interferometry（BLI）原理，该原理通过改变表面反射的光信号，实现对生物分子相互作用的监测。
BLI技术具有高灵敏度、快速响应和实时监测等优点，已广泛用于生物学研究、药物开发和临床诊断等领域。
应用领域Fortebio的技术广泛应用于生物领域的各个方面，包括但不限于以下应用领域：蛋白质相互作用研究Fortebio的技术可以实时监测和分析蛋白质之间的相互作用。
通过测量蛋白质的结合动力学和亲和力，研究人员可以深入了解蛋白质的功能和作用机制。
这对于药物开发、蛋白质工程和生物学研究都具有重要意义。
药物研发Fortebio的技术可以帮助加速药物研发过程。
通过实时监测药物与靶标蛋白的相互作用，研究人员可以评估药物的亲和力和效力，优化药物设计和筛选潜在药物候选物。
这有助于加快药物研发的速度和降低研发成本。
病原体检测与诊断Fortebio的技术还可以应用于病原体的检测和诊断。
通过与病原体相关的抗原或抗体的相互作用，可以提高病原体的检测灵敏度和特异性。
这对于传染病的快速诊断和筛选病原体也具有重要意义。
基因组学研究Fortebio的技术可以用于研究基因组学。
通过检测DNA的结合与修饰，研究人员可以了解基因的表达调控机制和细胞过程。
这对于基因组学研究和疾病诊断有重要意义。
BioLegend Matrix A-F 安全技术说明书
Matrix A-F 无资料。
固体。
化学品中文名称其他标识手段产品类型:::安全技术说明书根据 GB/ T 16483-2008 和 GB/ T 17519-2013化学品安全技术说明书Matrix A-F化学品的推荐用途和限制用途+1-858-455-9588 (7:00AM – 5:00PM PT, M-F)应急咨询电话（带值班时间）:供应商/ 制造商:BioLegend Inc.8999 BioLegend WaySan Diego, CA 92121 – USATel: +1-858-455-9588 (7:00AM – 5:00PM PT, M-F)产品用途:研究。
工业应用.应用范围:本安全技术说明书责任人的e-mail地址:Matrix A-F :Product name 产品代码:无资料。
GHS危险性类别:标签要素物质或混合物的分类根据 GB13690-2009 和 GB30000-2013H303急性毒性 (口服) - 类别 5H312急性毒性 (皮肤) - 类别 4H332急性毒性 (吸入) - 类别 4H402危害水生环境一急性危险 - 类别 3H412危害水生环境一长期危险 - 类别 3紧急情况概述警示词:警告危险性说明:H303 - 吞咽可能有害。
H312 + H332 - 皮肤接触或吸入有害。
H402 - 对水生生物有害。
H412 - 对水生生物有害并具有长期持续影响。
防范说明预防措施:P280 - 戴防护手套和穿防护服。
P271 - 只能在室外或通风良好之处使用。
P273 - 避免释放到环境中。
P261 - 避免吸入粉尘。
事故响应:P304 + P340, P312 - 如误吸入： 将受害人转移到空气新鲜处，保持呼吸舒适的休息姿势。
如感觉不适，呼叫解毒中心或医生。
P301 + P312 - 如误吞咽： 如感觉不适，呼叫解毒中心或医生。
P362 + P364 - 脱掉所有沾染的衣服，清洗后方可重新使用。
Fortebio生物分子相互作用检测平台-中文版
Microplate Formats板型
Wide Fit Within Discovery and Development Process
Research 研究
Screening 筛选
Lead Characterization
Preclinical and Clinical临床前
期及临床
Manufacturing and QC
生产及质控
Assay development 实验发现
> 150 D 2, 5, or 10 Hz
16 1 - 16
2 Yes Yes 96 / 96HA 384 / 384TW 40 µL per well
50 ng/ml to 2 mg/ml
1 mM to 10 pM 2, 3, or 6 pM
Fortebio Octet 检测平台
Affinity characterization
> 5000 D
0.3 Hz or 0.6 Hz 2
1 - 16 2 Yes Yes
96 / 96HA 384 / 384TW 40 µL per well
> 150 D 2, 5, or 10 Hz
8 1-8
1 No No
96
180 µL per well
100 ng/ml to 700 ug/ml
Expression Monitoring
Fortebio Octet 生物膜干涉技术(BLI)常见问题解答-第二版
生物膜干涉技术(BLI) 常见问题解答（第二版）目录缩写说明 (2)动力学相关术语 (2)传感器和仪器相关术语 (2)生化相关术语 (3)1 实验基本常识 (3)1.1如何自学 (3)1.2关于动力学 (3)1.3关于耗材与样品要求 (5)1.4关于生物膜干涉技术 (7)1.5软件与硬件性能参数 (8)1.6关于应用 (11)1.7关于基本操作 (11)2 动力学实验设计与操作 (15)2.1 关于固化和传感器的选择 (15)2.2 关于结合和解离 (19)2.3 关于传感器再生 (20)2.4 小分子检测 (21)2.5 动力学实验操作 (23)2.6 动力学实验数据处理 (29)2.7实验常见问题（动力学部分） (39)3 定量实验设计与操作 (44)3.1定量实验设计 (44)3.2 定量实验操作 (46)3.3定量实验数据处理 (48)3.4实验常见问题（定量部分） (51)4 仪器维护与硬件问题 (51)5 联系我们 (55)缩写说明动力学相关术语k on或k a：结合速率常数，单位M-1s-1k off或者k d：解离速率常数，单位s-1K D：亲和力常数（解离平衡常数），单位Mk obs：表观结合常数[C]：分析物浓度Ligand：配体，即固化在传感器上的物质Analyte：分析物，即与固化物质结合的物质R max：理论上分析物结合信号的最大值，即所有ligand均被analyte结合后所获得的信号Req：分析物结合达到平衡时的信号值Initial slope：起始斜率BLI：生物膜干涉技术Baseline：平衡步骤Association：结合步骤Dissociation：解离步骤Loading：固化步骤传感器和仪器相关术语SA：链霉亲和素传感器SSA：超级链霉亲和素传感器APS：氨基丙基硅烷传感器AR2G：氨基偶联传感器AHC：抗人免疫球蛋白Fc段传感器AHQ：人免疫球蛋白定量传感器AMQ：鼠免疫球蛋白定量传感器AMC：抗鼠免疫球蛋白Fc段传感器NTA：镍离子传感器anti-His：抗组氨酸标签传感器anti-GST：抗GST标签传感器Capture类传感器：除去SA，SAX，SSA，AR2G，APS外的传感器Octet：Octet家族仪器，包含Octet RED，RED96，QK，QKe，QK384，RED384，HTX，K2RED系列仪器：RED，RED96，RED384，K2QK系列仪器：QK，QKe，QK384生化相关术语EDC：1-(3-二甲基氨丙基) -3-乙基-碳化二亚胺NHS：N-羟基琥珀酰亚胺HRP：辣根过氧化物酶DAB：二氨基联苯胺，一种HRP的底物，在HRP催化后可以生成金属沉淀EMSA：电泳迁移率实验，用来研究DNA和蛋白的结合Epitope binning：表位分析His-tag：组氨酸标签Co-IP：免疫共沉淀PBS：磷酸盐缓冲液BSA：牛血清白蛋白hIgG：人免疫球蛋白mIgG：鼠免疫球蛋白1:1反应：一份的ligand与一份的analyte形成一份的产物，即两者只有一个结合位点2:1反应：Ligand上有2个可以和analyte的结合位点，并且两者独立1:2反应：Analyte上有2个可以和ligand结合的位点，并且两者独立1 实验基本常识1.2关于动力学1.2.1 k on, k off, K D值各代表什么意义？生物分子间的相互作用一般视为可逆反应。
QIAGEN Perfe CTa qPCR ToughMix UNG ROX 产品说明书
©2018 QIAGEN Beverly, Inc. 100 Cummings Center Suite 407J Beverly, MA 01915 Quantabio brand products are manufactured by QIAGEN Beverly, Inc.Intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.95139 / IFU-095.1 REV 02PerfeC T a ® qPCR ToughMix ® UNG ROX ™DescriptionPerfeC T a qPCR ToughMix UNG ROX is a 2X concentrated ready-to-use reaction cocktail for PCR amplification of DNA templates that overcomes many known inhibitors of PCR often present in crude samples extracted from environmental specimens, plant tissues, or animal tissues. It is a versatile and robust real-time qPCR reagent that provides maximum sensitivity and PCR efficiency with a variety of fluorogenic probe chemistries, including TaqMan ® hydrolysis probes. PerfeC T a qPCR ToughMix UNG ROX contains all required reaction components, except primers, probe(s), and DNA template. Inclusion of uracil DNA glycosylase (UNG), and substitution of dTTP with dUTP, prevents amplification of carry-over contamination from previous dU-containing PCRs.A key component of PerfeC T a qPCR ToughMix UNG ROX is an ultra pure, highly processive thermostable DNA polymerase that is combined with high avidity monoclonal antibodies. This proprietary polymerase mix is highly resistant to PCR inhibitors and provides an extremely stringent automatic hot-start allowing reaction assembly, and temporary storage, at room temperature prior to PCR amplification. PerfeC T a qPCR ToughMix UNG ROX delivers exceptional performance with either fast or conventional PCR cycling protocols.Instrument CompatibilityDifferent real-time PCR systems employ different strategies for the normalization of fluorescent signals and correction of well-to-well optical variations. It is important to match the appropriate reference dye to each specific optical detection system. PerfeCta qPCR ToughMix, ROX contains an optimal concentration of a stabilized carboxy-X-rhodamine compound (ROX ™) for instruments that use an excitation wavelength of ~490 nm and 605 to 610 nm emission channel for the reference signal. Please consult our Product Finder selection tool at to find the correct product for your real-time PCR system. ComponentsPerfeC T a qPCR ToughMix UNG ROX (2X): 2X concentrated reaction buffer containing optimized concentrations of MgCl 2, dNTPs (dATP, dCTP, dGTP,dUTP), hot-start DNA polymerase, uracil DNA glycosylase (UNG), and stabilizers.Storage and StabilityStore components in a constant temperature freezer at -25°C to -15°C protected from light upon receipt. Repeated freezing and thawing does not impair product performance. After thawing, mix thoroughly before using.For lot specific expiry date, refer to package label, Certificate of Analysis or Product Specification Form.Guidelines for qPCR:▪ The design of highly specific primers and probes is a critical parameter for successful real-time PCR. The use of computer aided primer design programs is encouraged in order to minimize the potential for internal secondary stru cture and complementation at 3’-ends within each primer, the primer pair, and primer/probe combinations. For best results, amplicon size should be limited to 65 - 200 bp. Optimal results may require titration of primer concentration between 100 and 900 nM. A final concentration of 300 - 400 nM each primer and 100 to 250 nM probe is effective for most applications. Increasing the concentration of the primer that initiates synthesis of the target strand that is complementary to the probe can improve fluorescent signal for some primer/probe systems.▪ Preparation of a reaction cocktail is recommended to reduce pipetting errors and maximize assay precision. Assemble the reaction cocktail with all required components except sample template (genomic DNA or cDNA) and dispense equal aliquots into each reaction tube. Add the DNA template to each reaction as the final step. Addition of samples as 2 to 5- L volumes will improve assay precision.▪ Suggested input quantities of template are: cDNA corresponding to 1 pg to 100 ng of total RNA; 10 pg to 1 µg genomic DNA▪ After sealing each reaction, vortex gently to mix contents. Centrifuge briefly to collect components at the bottom of the reaction tube. Reaction AssemblyComponentVolume for 20-μL rxn.Final ConcentrationPerfeC T a qPCR ToughMix UNG ROX (2X) 10 µL 1xForward primer variable 100 – 900 nM Reverse primer variable 100 – 900 nM Probevariable 100 – 250 nMNuclease-free water variable Template2 – 5 µL variable Final Volume (μL)20 µLNote : For smaller or larger reaction volumes scale all components proportionally.Cat No. 95139-250 Size: 250 x 20-uL reactions (2 x 1.25 mL) Store at -25ºC to - 15°C protected from light95139-012 1250 x 20-µL reactions (10 x 1.25 mL)95139-05K5000 x 20-µL reactions (1 x 50 mL)©2018 QIAGEN Beverly, Inc. 100 Cummings Center Suite 407J Beverly, MA 01915 Quantabio brand products are manufactured by QIAGEN Beverly, Inc.Intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.95139 / IFU-095.1 REV 02PCR Cycling ProtocolFast 2-Step Cycling Fast 3-Step Cycling Standard Cycling UNG carry-over incubation – optional:Initial denaturation: PCR cycling (30-45 cycles):The appropriate step for fluorescent data collection varies for different probe assay formats. Data collection for 5’-nuclease probe assays (TaqMan probe) should be carried out at the end of the extension step. Use the annealing step for data collection with hybridization probe assays (HybProbe ® FRET hybridization probes, Molecular Beacons, Solaris ® qPCR Assays, Scorpions ® primers, etc.).End-point analysis should be carried out at a suitable temperature for your detection probe chemistry.‡ UNG incubation is optional. Alternate protocols are acceptable. We find that a 5 minute incubation at 45ºC is significantly more effective at eliminating carry-over contamination than the more typical procedure of 50ºC for 2 min.* Full activation of the DNA polymerase occurs within 10 seconds at 95ºC; however, optimal initial denaturation time is template dependent and will affect qPCR efficiency and sensitivity. Amplification of genomic DNA or supercoiled plasmid DNA targets may require 5 to 10 min at 95ºC to fully denature and fragment the template. Short double-stranded DNA template (PCR product) or single-stranded DNA template, such as cDNA, may require as little as 1s at 95ºC. Use 30s at 95ºC as a general starting point.† Extension time is dependent upon amplicon length and the minimal data collection time requirement for your qPCR instrument. Use 30s at 60ºC as a general starting point. Some assay designs and/or detection chemistries may require a 3-step cycling protocol for optimal performance. Optimal annealing temperature and time may need to be empirically determined for any given primer set and real-time instrument.Quality ControlKit components are free of contaminating DNase and RNase. PerfeC T a qPCR ToughMix UNG ROX is functionally tested in qPCR. Kinetic analysis must demonstrate linear resolution over six orders of dynamic range (R 2 > 0.990) with a 2-fold discrimination of starting template and a PCR efficiency > 95%.Limited Label LicensesUse of this product signifies the agreement of any purchaser or user of the product to the following terms:1. The product may be used solely in accordance with the protocols provided with the product and this manual and for use with components contained in the kit only. QIAGEN Beverly, Inc. grants no license under any of its intellectual property to use or incorporated the enclosed components of this kit with any components not included within this kit except as described in the protocols provided with the product, this manual, and additional protocols available at Some of these additional protocols have been provided by Quantabio Product users for Quantabio users. These protocols have not been thoroughly tested or optimized by QIAGEN Beverly, Inc.. QIAGEN Beverly, Inc. neither guarantees them nor warrants that they do not infringe the rights of third-parties.2. Other than expressly stated licenses, QIAGEN Beverly, Inc. makes no warranty that this kit and/or its use(s) do not infringe the rights of third-parties.3. This kit and its components are licensed for one-time use and may not be reused, refurbished, or resold.4. QIAGEN Beverly, Inc. Specifically disclaims any other licenses, expressed or implied other than those expressly stated.5. The purchaser and user of the kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above. QIAGEN Beverly, Inc. may enforce the prohibitions of this Limited License Agreement in any Court, and shall recover all its investigative and Court costs, including attorney fees, in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the kit and/or its components.This product is licensed under U.S. Patent No. 7,972,828 and corresponding US and foreign patents and patent applications for any use for research and development purposes; the license expressly excludes any use for diagnostic testing or clinical therapeutics in humans or animals.The use of this product is covered by at least one claim of U.S. Patent No. 7,687,247 owned by Life Technologies Corporation. The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The buyer cannot sell or otherwise transfer (a) this product, (b) its components, or (c) materials made by the employment of this product or its components to a third party or otherwise use this product or its components or materials made by the employment of this product or its components for Commercial Purposes. Commercial Purposes means any activity for which a party receives or is due to receive consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. The buyer cannot use this product or its components or materials made using this product or its components for therapeutic, diagnostic or prophylactic purposes. Further information on purchasing licenses under the above patents may be obtained by contacting the Licensing Department, Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008. Email: *************************.PerfeC T a, and ToughMix are registered trademarks of QIAGEN Beverly, Inc.. TaqMan is a registered trademark of Roche Molecular Systems, Inc. HybProbe is a registered trademark of Roche Diagnostics GmbH. ROX is a trademark Life Technologies Corporation. Solaris is a registered trademark of Thermo Fisher Scientific Inc. Scorpions is a registered trademark of DxS, Ltd. of Manchester, UK.。
药物分子设计常用软件
可以安装在 windows，linux 等不同系统下，可以在 windows 下调用 NAMD 和 AMMP 等模拟软件。可以在图形化介面下轻易操作。当然，具体的参数还是要看 NAMD 自己的 manual 来弄清楚。这个软件是意大利人开发的，有个独特的功能，一个是他们支持一个叫 做 VEGA ZZ Live CD Creator 的安装模式，这样可以把这个软件放在一个 U 盘中带到哪台 机器都可以用。
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药 物 分 子设计常用软件
computer automatically via Internet for free. You can download and use locally PASS demo version, which predicts 50 biological
6. ArgusLab
A molecular modeling, graphics, and drug design program ArgusLab is freely licensed Over 20,000 downloads and still growing! 网址：/
中文名: InsightII 生物分子模拟软件 英文名: ACCELRYS INSIGHT II 2005 LINUX READ NFO-QUASAR 资源格式: 光盘镜像 制作发行: Accelrys 地区: 美国 语言：英语 日语 网址：/products/insight/index.html 类别：生物分子模拟
1） 核心模块及图形界面 2） 结构模型构建工具 3） 能量计算工具 4） 生物大分子结构模建与性质分析 5） 基于靶标结构的药物设计 6） 核磁共振结构测定
蛋白质功能-结构-相互作用预测网站工具合集
蛋白质组学蛋白质是生物体的重要组成部分，参与几乎所有生理和细胞代谢过程。
此外，与基因组学和转录组学比较，对一个细胞或组织中表达的所有蛋白质，及其修饰和相互作用的大规模研究称为蛋白质组学。
蛋白质组学通常被认为是在基因组学和转录组学之后，生物系统研究的下一步。
然而，蛋白质组的研究远比基因组学复杂，这是由于蛋白质内在的复杂特点，如蛋白质各种各样的翻译后修饰所决定的。
并且，研究基因组学的技术要比研究蛋白质组学的技术强得多，虽然在蛋白质组学研究中，质谱技术的研究已取得了一些进展。
尽管存在方法上的挑战，蛋白质组学正在迅速发展，并且对癌症的临床诊断和疾病治疗做出了重要贡献。
几项研究鉴定出了一些蛋白质在乳腺癌、卵巢癌、前列腺癌和食道癌中表达变化。
例如，通过蛋白质组学技术，人们可以在患者血液中明确鉴定出肿瘤标志物。
表1列出了更多的蛋白质组学技术用于研究癌症的例子。
另外，高尔基体功能复杂。
最新研究表明，它除了参与蛋白加工外，还能参与细胞分化及细胞间信号传导的过程，并在凋亡中扮演重要角色，其功能障碍也许和肿瘤的发生、发展有某种联系。
根据人类基因组研究，约1000多种人类高尔基体蛋白质中仅有500~600种得到了鉴定，建立一条关于高尔基体蛋白质组成的技术路线将有助于其功能的深入研究。
蛋白质组学是一种有效的研究方法，特别是随着亚细胞器蛋白质组学技术的迅猛发展，使高尔基体的全面研究变为可能。
因此研究人员希望能以胃癌细胞中的高尔基体为研究对象，通过亚细胞器蛋白质组学方法，建立胃癌细胞中高尔基体的蛋白质组方法学。
研究人员采用蔗糖密度梯度的超速离心方法分离纯化高尔基体，双向凝胶电泳（2-DE）分离高尔基体蛋白质，用ImageMaster 2D软件分析所得图谱，基质辅助激光解吸离子化飞行时间质谱（MALDI-TOF MS）鉴定蛋白质点等一系列亚细胞器蛋白质组学方法建立了胃癌细胞内高尔基体的蛋白图谱。
最后，人们根据分离出的纯度较高的高尔基体建立了分辨率和重复性均较好的双向电泳图谱，运用质谱技术鉴定出12个蛋白质，包括蛋白合成相关蛋白、膜融合蛋白、调节蛋白、凋亡相关蛋白、运输蛋白和细胞增殖分化相关蛋白。
Fortebio Octet BLI 蛋白生物素标记应用指南
蛋白的生物素标记操作指南（Octet分子互作仪器测定专用）概述链霉亲和素与生物素之间的相互作用是目前已知强度最高的非共价作用，并且二者的结合稳定性好，专一性强，不受试剂浓度，pH环境，抑或蛋白变性剂等有机溶剂影响。
因此，链霉亲和素与生物素快速，稳定和不可逆非共价的结合被广泛应用于研究生物分子间的相互作用。
Octet平台的链霉亲和素（SA/SAX/SSA）生物传感器已被开发用于蛋白定量和动力学，结合在SA//SAX/SSA传感器上的第一个蛋白质必须进行生物素化标记。
为方便利用Octet平台上的SA/SAX/SSA传感器进行动力学和定量分析，特制订本指南为利用生物素化试剂标记目标蛋白提供指导。
生物素试剂的选择Biotin为例。
生物素化试剂建议购买小包装，使用DMSO溶解配制成10 mM母液保存于-20℃备用。
产品货号：21312的NHS-PEG12-Biotin为例：10 mM母液的配制案例=（生物素化试剂质量1mg）÷（生物素分子量941 Da）×100=0.106 mL，1 mg生物素化试剂溶于0.106 mL DMSO中即为10 mM母液。
氨基偶联生物素化试剂反应示意图实验试剂和耗材1)缓冲液：DMSO，1×PBS，PBST（PBS+0.02% tween20）2)掌上离心机，一台3)生物素标记试剂EZ-Link NHS-PEG12-Biotin (Thermo，货号：21312) 1 mg，加入0.106 mL的DMSO配置成10 mM母液4)PD Min iTrap™ G-25 Desalting Column用于脱盐，蛋白样品体积0.1-0.5 mL，(GE, 货号：28-9180-07 )。
5)Zeba desalting spin columns脱盐柱：a)蛋白样品体积30-130 uL,使用0.5 mL脱盐柱(Thermo, 货号：89882)b)蛋白样品体积200-700 uL,使用2 mL脱盐柱(Thermo, 货号：89889)c)蛋白样品体积600-2000 uL,使用5 mL脱盐柱(Thermo, 货号：89891)6)透析装置：Slide-A-Lyzer™ Dialysis Cassettes（Thermo，具体型号根据目标蛋白样品体积以及目标蛋白的分子量进行选择。
FortBio Octet 培训使用章程
Application of white light interferometry in monitoring molecular interact
White light Constructive interference 100% Partially constructive interference
KD =
kd ka
R=R0+Req(1-e-kobs*t)
Kobs=Ka*[B]+Kd
Req为 Rt为 analyte浓
R=R0+Rte-kd*t
数
结 时 终 当
观结
11
传
Warm for 30min
Hydrate(prewet) for at least 10min
Prepare sample plate and warm it to RT.
Antibody Fragments
Proteins Peptides Nucleotides (DNA)
1 nm 1,000 150 1
Antigen – Fusion Protein Antibody - Peptide Multiple Antibody Pairings Antibody - Small Molecule
Cycle 4
40
Ligand or analyte?
One-sБайду номын сангаасot assay
36
Setting in processing data analysis
Agenda
OCTET
37
baseline
结
传 进 测试
38
Capture method: 传 结 传
ICH-GCP E6(R2)中文版
E6(R2)人用药品注册技术要求国际协调会ICH协调指导原则ICH指导委员会2016年11月9日当前版本：第四阶段中文编译：中国GCP联盟 & 临床研究大汇E6(R1)译者序公元1996年，ICH-GCP正式发布R1版，彼时之中国，了解GCP的人仅限于当时中国卫生部培养的数百名医学专家，规范的临床试验法规与体系还在起草中。
1998年3月卫生部而发布了中国第一部GCP（试行），同年5月实施；1998年国务院机构改革成立了国家药品监督管理局，1999年9月1日实施的《药品临床试验管理规范》（局令13号，已废止），在整整4年之后的2003年9月1日，我国的GCP，《药物临床试验质量管理规范》（局令第3号）颁布实施并持续至今。
受制于起步阶段的能力所限，我们的GCP法规只有70条款共计12998字，而对比ICH-GCP则有383条款共计27936单词。
R1版的ICH-GCP，2003年国家药监局中国药品生物制品检定所获权组织专家翻译，才有过中文版（未曾公开发布），陆续有过多个版本的企业/组织发布版本，而以国家药品审评中心（CDE）发布的官译稿件，时间却很明确，是在2016年8月5日才得以发布（如下图）。
可以说，无论是标准的水平高低还是时代的步伐快慢，我们都曾落后了太多太多。
人生如梦，岁月如歌，春去秋来，夏行冬至。
二十载岁月匆匆而过。
2016年11月30日，ICH正式颁布了GCP的增补件R2，标志着全球药物临床试验进入到了一个崭新的时代，无独有偶，仅仅过去了2天，2016年12月2日，中国国家食品药品监督管理总局发布了《药物临床试验质量管理规范》的第二次征求意见稿，大量新内容参考了ICH-GCP R1甚至R2，意见稿直接以超30000字的信息量向全中国全世界展现它的雄心：今天的和未来的中国药物临床试验，在经历了蹒跚学步与青春期的躁动之后，正大幅度的向着国际先进水平看齐。
天行健，君子当自强不息，“中关村玖泰药物临床试验技术创新联盟/中国药物临床试验机构联盟”携手“临床研究大汇”，有志于协助我国临床研究行业在这次革新中及时跟上时代潮流，我们在R2发布后的7日内，参考借鉴CDE的R1中文版，完成了中文版的翻译与校对工作，在此时正式向全球华语用户推送，由于时间紧，我们的工作可能有不足之处，在此虚心并诚恳的接受所有批评意见。
ForteBio Octet RED96e 生物分析仪说明书
Key features• High-quality kinetic screening and affinity characterization • Microfluidics-free Dip and Read™ format reduces assay time and maintenance cost• Eight parallel, independent channels for maximum speed and flexibility• Versatility to detect anything from small molecules to mammalian cells• Non-destructive sampling allows full sample recovery• Up to 12 hours of unattended run time• Sample plate cooling for temperature sensitive proteins• Perfectly suited to operate in GxP-regulated environmentsThe Octet® RED96e system detects a diverse range of bio-molecules from small molecules to proteins to mammalian cells. The Octet platform offers an advanced fluidics-free approach with a wide variety of off-the-shelf Dip and Read biosensors for rapid binding kinetics and quantitation analysis. The system utilizes ForteBio’s Bio-Layer Interferometry (BLI) technology, enabling direct detection of specific proteins and other biomolecules — even in complex mixtures like cell cul-ture supernatants and lysates. The 8-channel Octet RED96e system performs quantitation of 96 samples in 32 minutes, and kinetic screening of 64 samples in 1.5 hours. Analysis can be done using a single channel or up to eight channels, enabling more flexibility in sample throughput when needschange. An optional microplate evaporation cover minimizes losses in sample volume, allowing full post-analysis sample recovery even after a 12 hour experiment.Figure 1: Human IgG Quantitation. Example data from human IgG analyte binding to Protein A biosensors. Binding was performed at 30°C, with a shake speed of 1000 rpm and a two-minute read per well. Human IgG solution was prepared at 0.025 μg/mL up to 300 μg/mL and the standard curve shown on a log-log scale was generated using the initial slope algorithm and fitted with the unweighted 5-parameter logistic (5PL)regression model.BindingRateConcentration (µg/mL)1010101010Quantitation assaysThe Octet RED96e system directly measures the presence of specific proteins and other molecules in solution with minimal interference from complex matrices. Accurate and reproducible concentrations can be determined in as little as two minutes per sample or 32 minutes for a whole plate using a simple, one-step assay (Figure 1). High sensitivity in quantitation can be achieved to sub-ng/ml levels with 2-step and 3-step assay formats, allow-ing automated measurement of contaminants such as host cell proteins and residual protein A faster and more precisely than ELISA. Process economics can be improved further by regener-ating and re-using the biosensors.Octet RED96e SystemUnmatched versatility for discovery, development and quality controlKinetic assaysThe Octet RED96e system monitors binding events in real time to calculate on rates (k a ), off rates (k d ), and affinity con-stants (K D ). The superior sensitivity of the system enables measurement of small organic molecules (Figure 2) and kinetic constants over a broad range. The temperature of one 96-well sample plate can be controlled from 15–40°C, which enables reliable kinetic determinations from low up to physiological temperatures for temperature sensitive proteins (Figure 3). Additional advantages afforded by sample cooling include the ability to rapidly determine binding rate constants at multiple temperatures to extrapolate thermodynamic mea-surements. The Octet RED96e system’s eight channels can be used independently to measure samples for screening pur-poses or in tandem, pairing the sample read with a dedicated reference for high-quality kinetic characterization.B i n d i n g (n m )20040060080010001200Time (sec)00.050.100.150.200.25A4.88E-092.85E+041.39E-04Time (sec)B i n d i n g (n m )200400600800100012000.050.150.250.350.45Bk d (1/s)5.64E-094.59E+042.59E-04Time (sec)B i n d i n g (n m )2004006008001000120000.050.150.250.350.450.55CK D (M)k a (1/Ms)k d (1/s)6.73E-095.30E+043.57E-04B i n d i n g (n m )Time (sec)K D k a k d 2.00E-61.53E53.06E-1Octet Data Analysis HT softwarePre-defined templates in Octet Data Acquisition softwarestreamlines setup prior to running an assay and minimizes train-ing needs. Octet Data Analysis High Throughput (HT) software can overlay data from multiple plates over an extensive range of parameters and metrics to analyze acquired data from an entire project, thereby reducing analysis time from hours to minutes. Data Analysis HT Analysis settings in Octet DataAnalysis HT software can be saved and re-loaded for new simi-lar datasets to speed up routine assays. The software can also generate customized reports of the experiments, combining various data elements such as graphs, text boxes, data tables, images and experimental details (Figure 4). These reports are ready to be uploaded to an electronic notebook or stored in the company database.Figure 2: Small molecule kinetics. Example data from benzenesulfonamide(MW 157 Da) binding to biotin-carbonic anhydrase loaded on Super Streptavidin biosensors. Binding was performed at 25°C, with a shake speed of 1000 rpm. A 100 μM benzensulfonamide solution was prepared and serially diluted 1:4.Figure 3: Large molecule characterization. Example data from human Prostate Specific Antigen (PSA, MW 30 kDa) binding to a biotinylated anti-human PSA mouse monoclonal antibody loaded onto Streptavidin biosensors. Binding was performed at 15°C (A), 25°C (B) and 30°C (C), with a shake speed of 1000 rpm. A 200 nM PSA solution was prepared and serial diluted 1:2 to obtain the 5 concentrations run.Figure 4: Octet Data Analysis HT software enables making customized reports that can be uploaded into electronic notebooks and added to the database. In addition to customized report, Data Analysis HT enables analysis of multiple plates and experiments together to maximize workflow efficiency.Operate in GxP regulated environmentsThe Octet RED96e system has been developed to operate reli-ably in a regulated environment. ForteBio offers 21 CFR Part 11 software and a full line of GxP products and services as part of the Octet RED96e GxP Package. These include:• Octet CFR software and ForteBio FB Server features such as:•Controlled access with multiple user privileges — adminis-trator, developer, supervisor, lab user•Primary data integrity — digitally signed acquired data thatis rendered invalid after data tampering•Electronic signatures — enable data to be locked afteranalysis is complete•Enhanced audit trail — all actions are recorded and time-stamped with details of old vs. new values•Full control of routine assays that speed up analysis— method files and analysis settings can be saved forroutine assays•Customized reports — created by combining various dataelements such as graphs, text, data tables and imagesready to be uploaded to your ELN• Installation and Operational Qualification (IQ/OQ) and Per-formance Qualification (PQ) packages ensure your systemis qualified and operate as intended and that performance meets specifications• Performance Certification (PC) services maintain your system in a calibrated state and in peak condition• Customer-run Software Validation Package and support to trim validation time down to just three days• Biosensor Validation Support Services for multiple biosensor lot sampling and selection• Excellent Global T echnical Support assistance Octet RED96e system specifications*DetectiontechnologyBio-Layer Interferometry (BLI)Biosensor type Disposable, single-use fiber optic biosensorswith optional reuse by regeneration and/orre-racking in the sensor tray Informationprovided• Yes/No binding• Kinetic and affinity analysis (k obs, k a, k d, K D)• Specific and selective detection ofmolecules, even in crude samples• Relative and absolute quantitation ofspecific proteins in crude matrices orpurified samplesData presentation• Plots displaying real-time kinetic bindingsensorgrams, fitted result plots, andresiduals of fits• Concentration data analysis includingcalibration curves and output of tabulatedconcentration data• Tabulated kinetic data• Epitope binning and cross-blockingmatrices and trace overlaysSample types Proteins, antibodies, peptides, DNA, RNA,liposomes, bacterial cells, viruses, mamma-lian cells, small molecules in various mediaincluding serum, buffers containing DMSO,periplasmic fractions, untreated cell culturesupernatants, and crude cell lysates Number ofspectrometers8Maximumsimultaneous reads8Data collection rate2, 5, or 10 HzSample positionand format1 standard 96-well, black, flat bottommicroplateSample volume 180–220 μL/well, non-destructive testing Orbital flowcapacityStatic or 100–1500 rpmAnalysistemperature range15–40°C, 1°C increments* A ll specifications are subject to change without notice.ForteBio47661 Fremont Boulevard Fremont, CA 94538888.OCTET-75 or 650.322.1360************************ForteBio Analytics (Shanghai) Co., Ltd. No. 88 Shang Ke Road Zhangjiang Hi-tech Park Shanghai, China 201210*************************Molecular Devices (UK) Ltd. 660-665 Eskdale Winnersh Triangle Wokingham, Berkshire RG41 5TS, United Kingdom +44 118 944 8000*************Molecular Devices (Germany) GmbH Bismarckring 3988400 Biberach an der Riss Germany+ 00800 665 32860©2019 Molecular Devices, LLC. All trademarks used herein are the property of Molecular Devices, LLC. Specifications subject to change without notice. Patents: /product patents. FOR RESEARCH USE ONL Y. NOT FOR USE IN DIAGNOSTIC PROCEDURES.DS-41-0257 Rev CSafety standardsCE, NemkoOrdering informationPart No. UOM DescriptionOCTET RED96ESystemIncludes Octet RED96e instru-ment, Octet software, desktop computer, LCD monitor, acces-sory kit and one-year warranty OCTET RED96E-GxP SystemIncluded Octet RED96e instru-ment, Octet CFR software, desktop computer, LC monitor, accessory kit, IQ/OQ kit, PQ Kits and one-year warranty 18-5132PackSingle-use evaporation covers to extend the experiment up to 12 hours. 3 covers per packFor more information about ForteBio’s Octet platform for label-free, real-time detection of biomolecular interactions, applications, and services, visit or contact us directly.Workflow Up to 8 assays in parallel; up to 96 assays per 96-well plateAnalysis time per sampleHuman IgG quantitation in 2 minutes for 8 samples, ≤ 32 minutes for 96 samples Direct quantitation range for human IgG with Protein A Biosensor0.05–2000 μg/mL。
Fortebio Octet BLI数据分析-小分子动力学-1
The being detected thing is small molecular, and the sensor is immobilized with macromolecular. The detected way is by column. For example , the bio-protein A with IgG.1.双击数据分析软件图标，打开数据分析软件，在软件Data Selection界面左下侧子窗口路径下选中待分析kinetics数据：2.双击后载入左上侧子窗口Loaded Data Kinetics文件夹下3.左键单击所载入待分析数据，右侧窗口显示Sensor Tray按钮下界面，即该检测中sensor所在sensor tray上位置，在此可选择分析哪些sensor采集的数据，本例分析所有sensor的数据等。
5.选择所需分析sensor后，点击Processing进行数据处理：6.数据处理界面如下，该窗口左侧依次包括Step1:Data Selection，Step2:Subtraction，Step3:Align Y Axis，Step4:Inter-step Correction，Step5:Process，Step6:View Results，Step7: Save Results；右侧窗口默认为Raw Data View下界面，即原始数据预览。
7.点击Step 1下Sensor Selection按钮，界面如下：8.对于非经典小分子实验，在实验设计上包括sensor对照和孔对照（间经典小分子实验设计SOP），与经典小分子实验相比其差别在于浓度梯度按列排列，1个浓度1根sensor（经典小分子实验浓度梯度安行排列，所有浓度采用同一根sensor从低到高顺序检测）；数据处理时需分别扣除对照孔及对照sensor引入的干扰；选中对照孔，右键下选择Change Well Type…下的Refference Well：设置Refference Well后界面如下：9.在Sensor Tray下选中对照Sensor，右键选择Change Sensor Type…下的Refference Sensor：设置Refference Sensor后的界面如下：10.在Step 2下选中Double Subtraction，即扣除对照空及对照sensor可能引入的干扰：。
各类软件及其用途文档
三维分子类RASMOL 2.7.2.1 观看生物分子3D微观立体结构的软件。
非常有名，巨棒！RasTop 2.0 为RasMol 2.7.1的图形用户界面软件CHIME 2.6 SP3 直接在浏览器中观看3D分子。
MolMol 2k.1 将pdb等格式的蛋白文件通过微调，存成普通的图形文件。
CrystInfo 1.0 用来快速、容易地构建、观察与检查晶体3d结构。
PDViewer PDB格式文件的查看程序。
Weblab Viewlite 4.2 3维分子浏览工具及大量分子文件例子。
Weblab ViewerPro 4.2 Demo 3维分子浏览工具。
ICMLite 2.8 3维分子浏览工具，有一些其他软件没有的功能。
VMD 1.72 3维分子浏览工具，可以进行动态显示。
CN3D 3.0 3D分子结构观察软件。
WPDB 2.2 PDB文件检索显示分析软件。
DTMM 4.0 3维分子模型显示、编辑与构建程序。
Mole 1.1.8 Demo 高性能的大分子3维图形显示计算工具。
gopenmol 2.1 显示并分析分子结构及其特性的软件。
POV-Ray 3.5 beta rc5 生成三维图像工具软件。
WinMegaPov 0.7 3D渲染软件POV-Ray非官方编译软件。
MolPOV 2.0.8 将PDB文件转化为POV格式文件的软件。
Mol2Mol Demo 4.1 分子文件格式转换软件。
PovChem 2.1.1 将PDB文件转化为POV格式文件的软件。
Ortep-3 for Windows 1.074 生成分子的热椭圆形点图软件。
PLATON(2002.5.16版) 通用结晶学软件工具。
Mage 6.02 读取并演示Kinemage格式文件的专用软件。
Prekin 6.02 将PDB格式文件转换为Kinemage格式文件的软件。
Swiss-PdbViewer 3.7 PDB文件显示与分析软件。
DINAMO 蛋白序列排队比较编辑与三维模型构建工具软件。
Fortebio生物分子相互作用检测平台-中文版
Intensity λ =
ƒ(λ, ℓ)
Biolayer Interferometry（BLI）技术的原理
可实时检测到两个反射表面间距的改变
100% Relative Intensity 0
Distance between the two reflecting
nm shift
Wavelength (nm)
Octet Solution
ForteBio Inc.—艾瑞生物技术（上海）有限公司
• 公司的历史：
• • 2004年成立，在美国加州Menlo Park, 英国London, 和中国 上海建有生产和技术中心
• 独到的技术：
• • • • • • • • 新一代的label-free technology 生物膜干涉技术BioLayer Interferometry Technology（ BLI ）
buffer、预湿试剂、洗涤试剂分 别放置在不同的微孔板中
- 384样品板利用率100%
- 两个板位可独立使用96-plate或 384plate
Octet 系统与工作站联用
• Automated door and plate stage • Works with articulated arm and track-style robotic systems • Plate stage presents the biosensor tray, sample and reagent plates for robot • Octet software has automation hooks enabled to interact with robot software
• 皮克到毫克级的动态范围 污染物检测
Fortebio 分子相互作用仪
Octet 产品简介
Octet Instruments • 5种型号 - Octet QK, Octet Qke, Octet RED, Octet QK384, Octet RED 384 • 使用通用的样品板(96 and 384 well) • 没有流路系统 • 样品经过检测后，仍然留在原处，可以回收再使用
96, 96 HA
80 µL per well
0.1 mM to 0.1 nM
Octet QKe
Octet QK384 Octet RED96
> 5000 D 0.3 Hz or 0.6 Hz
1 1-8
1 Yes No
96, 96 HA
> 5000 D
0.3 Hz or 0.6 Hz 2
1 - 16 2 Yes Yes
Octet 软件
• 可灵活输出各种格式， Excel表，JEPG格式等 • 动力学分析直接得到ka, kd and KD 结果 • 浓度分析直接得到，ug/ml, ng/ml等浓度计算
Microplate Formats
Minimum Sample Volume
Affinity range (approximate) Sample Usage Temperature Control 21 CFR Part 11 Compliance
Octet QK
> 10,000 D 0.6 Hz 1 1-8 1 No No
Octet生物分子相互作用系统
ForteBio Inc.
• 2004年由华人物理学家创立, 2009年开始在中国销售 • 专利的生物膜干涉 Bio-Layer Interferometry (BLI)技术 • 总部在美国硅谷 • 全球超过350个用户，中国14个用户 • 全球第二的专业非标记技术厂商
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- 两个板位可独立使用96-plate或 384plate
Octet 系统与工作站联用
• Automated door and plate stage • Works with articulated arm and
track-style robotic systems • Plate stage presents the biosensor
/wiki/Interference
Biolayer Interferometry（BLI）技术的原理
改变膜厚度对干涉光的影响
100% 0
Wavelength
Relative Intensity
Biolayer Interferometry（BLI）技术的原理
实时无标记生物分子相互作用检测平台
Fortebio Octet 检测平台
ForteBio Octet QK、Qke、RED平台内部
Fortebio Octet 384全自动检测平台
• 使用96或384不同的微孔板类型 • 通量更高，样品消耗更少； • 384 tilted well 使用384微孔板，反应体系更小；
tray, sample and reagent plates for robot • Octet software has automation hooks enabled to interact with robot software
Octet 检测平台型号差异
Molecular Weight Range
Octet Solution
ForteBio Inc.—艾瑞生物技术（上海）有限公司
• 公司的历史：
• 2004年成立，在美国加州Menlo Park, 英国London, 和中国 • 上海建有生产和技术中心
• 独到的技术：
• 新一代的label-free technology • 生物膜干涉技术BioLayer Interferometry Technology（ BLI ）
Fortebio Octet 检测平台
Octet RED384/QK384 平台
Sensor 位置 自动舱门
样品板位置 反应板位置
31.5”
Biosensor 回收槽 29”
31.4”
Fortebio Octet 检测平台
ForteBio Octet QK384、RED384平台内部 - 双位置摆可将样品、缓冲溶液 buffer、预湿试剂、洗涤试剂分 别放置在不同的微孔板中
• Biocompatible Matrix
- minimizes non-specific binding
具有生物亲和性的表面基团阵列 极小的非特异性结合
• Uniform 一致性高
• Non-denaturing 不会使结合上的蛋白或抗体变性
Biolayer Interferometry（BLI）技术的原理
0
Wavelength (nm)
ƒ( ) Intensity λ = λ, ℓ
Biolayer Interferometry（BLI）技术的原理
可实时检测到两个反射表面间距的改变
100%
Relative Inten the two reflecting
ℓ surfaces =
两个反射表面间距的改变对干涉光强度的影响
Relative Intensity
Internal Reference (reflection surface 1)
100%
Distance between the two reflecting
ℓ surfaces =
Interface with Liquid (reflection surface 2)
生物分子结合后的 BLI Biosensor
任何分子的结合和解离都会引起不同波长光的干涉光强 度变化
Fortebio Octet 检测平台
Octet检测平台产品系列
• Octet Qk • Octet QKe • Octet QK384 • Octet RED • Octet RED384
Fortebio Octet 检测平台
Biolayer Interferometry（BLI）技术的原理
什么是光干涉现象
Biolayer Interferometry（BLI）技术的原理
干涉作用中的相互加强与相互抵消
a b
Two waves in phase Constructive interference
Two waves 180° out of phase Destructive Interference
ƒ( ) Intensity λ = λ, ℓ
nm shift
Wavelength (nm) Time
Biolayer Interferometry（BLI）技术的原理
特殊的 BLI Biosensor
• Two-dimensional binding surface 结合在二维的表面
|---- 600 µm ----|
• 广泛的应用： • 接近300台仪器 在全球各地广泛使用 • 生产并销售了超过2,500,000 个Biosensors
• 广阔的前景：
• 2nd in label-free detection system market share
•
HTStec March 2008 survey
/wiki/Interference
Data Acquisition Rate数据 # Spectrometers光通道 # Channels per Read 通道 Microplate Positions板位 Biosensor Reracking可回收 Robot Compatible兼容性
• 16通道检测，速度更快，时间更短 • QK384: 2 个独立光谱分析检测器，每8通道共用一个; • RED384: 16个独立光谱分析检测器，每个通道独立使用一个；
• 增加Biosensors 回放功能 • 有效提高biosensor使用率； • 更加节省耗材；
• 可与多种工作站联用提高自动化程度 • 软件兼容多种类型工作站；