Fortebio Octet定量简明指南

Small Molecule Kinetics Assay:概述：Octet可检测分子量为150 Da~1000 Da的小分子与蛋白间相互作用。

该SOP的目的在于为小分子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。

此外，该SOP假定所有使用者已经过ForteBio应用科学家上机培训，如若不然，请在培训完成后参考该SOP。

考量及建议：1.检测小分子时，应当使用SSA sensor（Cat# 18-0008 and 18-0009），该sensor是ForteBio公司目前唯一具备检测小分子灵敏度的传感器。

2.用于与小分子结合的蛋白或抗体必须先进行生物素化。

请参阅ForteBio技术说明书。

3.建议assay buffer为PBS+0.1%BSA+0.02%TW20+5%DMSO（如果为水溶性小分子，则无需加DMSO等有机溶剂）；4.设置protocol时，须设置两组SSA sensor，运行中第1组为实际检测用sensor，第2组为生物胞素（biocytin）封闭后、用作阴性对照的sensor。

5.检测sensor与对照sensor间运行步骤数以及各步运行时间必须一致——任何偏差将导致数据无法分析。

6.用于loding的生物素化蛋白浓度建议采用50ug/ml。

如蛋白很小或容易产生空间位阻，可能需要进行定量。

如蛋白较宝贵，其用量可以更低，但随后的结合效率可能会有所折扣（17kDa的蛋白在推荐浓度下3min后可产生2nm的shift）。

蛋白生物素化7.如果需要有机溶剂溶解小分子，稀释样品时DMSO最终浓度建议保持在5%以内（例如：溶于100%DMSO中20mM的样品，按照1:20溶于PBS后其浓度为1mM，DMSO为5%）。

8.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（或者2被剃度稀释）获取高度可信的Kd。

B RIEFINGFenofibrate Tablets. Because there is no existing USP monograph for this dosage form, a new monograph, based on validated methods of analyses, is proposed. The HPLCprocedures in the Assay and in the test for Organic Impurities are based on analysesperformed with LiChrosorb RP-18 brand of L1 column. The Synergi Hydro RP brand of L1 column is also suitable. The typical retention time of fenofibrate is about 9 min for columns with a 4.0-mm diameter and about 13–15 min for columns with a 4.6-mm diameter.The HPLC procedure in Dissolution Test 1 is based on analysis performed with Luna C18 brand of L1 column. The typical retention time for fenofibrate is about 0.6 min. The HPLC procedure in Dissolution Test 2 is based on analysis performed with the Phenomenex Prodigy ODS(3) brand of L1 column. The typical retention time for fenofibrate is about 10 min.(SM3: E. Gonikberg, L. Santos.)Correspondence Number—C99095; C104502Comment deadline: November 30, 2011Add the following:Fenofibrate TabletsDEFINITIONFenofibrate Tablets contain NLT 90.0% and NMT 110.0% of the labeled amount offenofibrate (C20H21ClO4).IDENTIFICATION• A.The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.ASSAY• P ROCEDUREAcidified water: Adjust the pH of water with phosphoric acid to 2.5 ± 0.1.Mobile phase: Acetonitrile and Acidified water (70:30)System suitability stock solution: 0.1 mg/mL each of USP Fenofibrate Related Compound A RS and USP Fenofibrate Related Compound B RS in acetonitrile.System suitability solution: 0.5 µg/mL each of USP Fenofibrate Related Compound A RS and USP Fenofibrate Related Compound B RS in Mobile phase from System suitability stock solutionStandard solution: 0.05 mg/mL of USP Fenofibrate RS in Mobile phaseSample stock solution: Prepare a solution containing approximately 2–4 mg/mL of fenofibrate by disintegrating the appropriate number of Tablets with sonication in Acidified water, using 30% of the final volume of the flask. Add acetonitrile to approximately 90% of flask volume, and sonicate with periodic swirling. Dilute with acetonitrile to volume. Sample solution: 0.05 mg/mL of fenofibrate in Mobile phase, based on the label claim from the Sample stock solution. Filter a portion of this solution, discarding the first few mL of the filtrate.Chromatographic system(See Chromatography 621, System Suitability.)Mode: LCDetector: UV 286 nmColumn: 4.0-mm × 25-cm or 4.6-mm × 25-cm; 5-µm or 4-µm packing L1Column temperature: 35Flow rate: 1.2 mL/minInjection size: 10 µLSystem suitabilitySamples: System suitability solution and Standard solutionSuitability requirementsResolution: NLT 2.0 between fenofibrate related compound A and fenofibrate related compound B peaks, System suitability solutionRelative standard deviation: NMT 2.0%, Standard solutionAnalysisSamples: Standard solution and Sample solutionCalculate the percentage of the labeled amount of fenofibrate (C20H21ClO4) in the portion of Tablets taken:Result = (r U/r S) × (C S/C U) × 100r U= peak response from the Sample solutionr S= peak response from the Standard solutionC S = concentration of USP Fenofibrate RS in the Standard solution (mg/mL)C U = nominal concentration of fenofibrate in the Sample solution (mg/mL)Acceptance criteria: 90.0%–110.0%PERFORMANCE TESTS• D ISSOLUTION 711Test 1Medium: 0.025 M sodium dodecyl sulfate in water; 1000 mLApparatus 2: 50 rpmTime: 30 minAcidified water: Adjust the pH of water with phosphoric acid to 2.5 ± 0.1.Mobile phase: Acetonitrile and Acidified water (70:30)Standard stock solution: 2.5 mg/mL of USP Fenofibrate RS in acetonitrile.Standard solution: Dilute the Standard stock solution with Medium to obtain a finalconcentration of about (0.001 ×L) mg/mL, where L is the label claim, in mg/TabletSample solution: Pass a portion of the solution under test through a suitable filter of 0.45-µm pore size, discarding the first few mL of the filtrate.Chromatographic system(See Chromatography 621, System Suitability.)Mode: LCDetector: UV 286 nmColumn: 2-mm × 3-cm; 3-µm packing L1Column temperature: 35Flow rate: 1.2 mL/minInjection size: 10 µLSystem suitabilitySample: Standard solutionSuitability requirementsTailing factor: NLT 0.9 and NMT 1.5Relative standard deviation: NMT 2.0%AnalysisSamples: Standard solution and Sample solutionCalculate the percentage of the labeled amount of fenofibrate (C20H21ClO4) dissolved:Result = (r U/r S) × (C S/L) ×V × 100r U= peak response from the Sample solutionr S= peak response from the Standard solutionC S = concentration of the Standard solution (mg/mL)L= label claim (mg/Tablet)V= volume of Medium, 1000 mLTolerances: NLT 80% (Q) of the labeled amount of fenofibrate (C20H21ClO4) is dissolved. Test 2: If the product complies with this test, the labeling indicates that it meets USP Dissolution Test 2.Medium: 0.05 M sodium dodecyl sulfate in water; 1000 mLApparatus 2: 50 rpmTime: 30 minBuffer: 136 mg/L of monobasic potassium phosphate in water. Adjust with diluted phosphoric acid to a pH of 2.9 ± 0.05.Mobile phase: Methanol and Buffer (80:20)Sample solution: Pass a portion of the solution under test through a suitable filter of 0.45-µm pore size, discarding the first few mL of the filtrate.Standard solution: (0.001 ×L) mg/mL of USP Fenofibrate RS in Mobile phase, where L is the label claim, in mg/TabletChromatographic system(See Chromatography 621, System Suitability.)Mode: LCDetector: UV 286 nmColumn: 4.6-mm × 15-cm; 5-µm packing L1Flow rate: 1.0 mL/minInjection size: 10 µLSystem suitabilitySample: Standard solutionSuitability requirementsTailing factor: NMT 2.0Relative standard deviation: NMT 2.0%AnalysisSamples: Standard solution and Sample solutionCalculate the percentage of the labeled amount of fenofibrate (C20H21ClO4) dissolved:Result = (r U/r S) × (C S/L) ×V × 100r U= peak response from the Sample solutionr S= peak response from the Standard solutionC S = concentration of the Standard solution (mg/mL)L= label claim (mg/Tablet)V= volume of Medium, 1000 mLTolerances: NLT 80% (Q) of the labeled amount of fenofibrate is dissolved.• U NIFORMITY OF D OSAGE U NITS 905:Meet the requirementsIMPURITIES• O RGANIC I MPURITIESAcidified water, Mobile phase, System suitability solution, Sample stock solution, andChromatographic system: Proceed as directed in the Assay.Standard solution: 0.5 µg/mL of USP Fenofibrate RS in Mobile phaseSample solution: 0.5 mg/mL of fenofibrate in Mobile phase, based on the label claim from the Sample stock solution. Filter a portion of this solution, discarding first few mL of filtrate.System suitabilitySamples: System suitability solution and Standard solutionSuitability requirementsResolution: NLT 2.0 between fenofibrate related compound A and fenofibrate relatedcompound B peaks, System suitability solutionRelative standard deviation: NMT 5.0%, Standard solutionAnalysisSamples: Standard solution and Sample solutionCalculate the percentage of each impurity in the portion of Tablets taken:Result = (r U/r S) × (C S/C U) × (1/F) × 100r U= peak response of each impurity from the Sample solutionr S= peak response of fenofibrate from the Standard solutionC S = concentration of USP Fenofibrate RS in the Standard solution (mg/mL) C U = nominal concentration of fenofibrate in the Sample solution (mg/mL) F= relative response factor (see Table 1)Acceptance criteria: See Table 1.Table 1Name RelativeRetentionTimeRelativeResponseFactorAcceptanceCriteria,NMT (%)Fenofibrate related compound A 0.34 1.3 0.2 Fenofibrate related compound B 0.36 1.0 0.2 (3RS)-3-[4-(4-Chlorobenzoyl)phenoxy]butan-2-one 0.50——aMethyl 2-[4-(4-chlorobenzoyl)phenoxy]-2-methyl-propanoate 0.65——aEthyl 2-[4-(4-chlorobenzoyl)phenoxy]-2-methyl-propanoate 0.80——a(4-Chlorophenyl)[4-(1-methylethoxy)phenyl]methanone 0.85——a Fenofibrate 1.00 ——Fenofibrate related compound C b 1.35 ——a Any unspecified impurity — 1.0 0.1Total impurities (includes fenofibrate related compounds A and B, and unspecified impurities) ——0.3a Disregard this impurity. It is a process impurity and is controlled in the drugsubstance monograph.b1-Methylethyl2-[[2-[4-(4-chlorobenzoyl)phenoxy]-2-methylpropanoyl]oxy]-2-methylpropano ate.ADDITIONAL REQUIREMENTS• P ACKAGING AND S TORAGE:Preserve in well-closed containers, and store at controlled room temperature.• L ABELING:When more than one Dissolution test is given, the labeling states the Dissolution test used only if Test 1 is not used.• USP R EFERENCE S TANDARDS 11USP Fenofibrate RSUSP Fenofibrate Related Compound A RS(4-Chlorophenyl)(4-hydroxyphenyl)methanone. C13H9ClO2 232.66USP Fenofibrate Related Compound B RS2-[4-(4-Chlorobenzoyl)phenoxy]-2-methylpropanoic acid, or fenofibricacid. C17H15ClO4 318.752S (USP35)。

溶液浓度 10g/100ml 溶液制备 10g/100ml 1g/100ml 0.5g/50ml 0.25g/25ml 0.1g/10ml 1g/1000ml 1g/1000ml 0.5g/500ml 0.25g/25ml 100mg/100ml 50mg/50ml 10mg/10ml 0.1g/1000ml 100g/1000ml 50g/500ml 25g/250ml 10g/100ml 5g/50ml 1g/10ml 0.01g/1000ml 10g/1000ml 5mg/500ml 1mg/100ml 相对不确定度（%） 称量 <0.01 0.02 0.04 0.08 0.02 0.02 0.04 0.08 0.2 0.4 2.0 0.2 0.4 0.8 2.0 4.0 20.0 2.0 4.0 20.0 体积 0.05 0.12 0.17 0.23 0.50 0.05 0.07 0.23 0.12 0.17 0.50 0.05 0.07 0.08 0.12 0.17 0.50 0.05 0.07 0.12 总不确定度 0.05 0.12 0.17 0.24 0.54 0.05 0.08 0.24 0.23 0.43 2.06 0.21 0.41 0.80 2.0 4.0 20.0 2.0 4.0 20.0
欧至关重要,应根据相对误差(允许误差除以标示体积)从常用玻璃仪器中 选用适当的移液管和容量瓶并制定最优的稀释方法(按照常用公式:每步稀释的相对误差平方之 和的平方根作为对照溶液稀释的相对误差的估计). 根据文献中玻璃仪器的容量公差限度标准,在给定的稀释比条件下,表 2 给出了最佳的稀释次数 和稀释效果.相关指南详见表 2(需要注意的是,这些因素当中不包括计数误差). 表 1 制备分析用供试品溶液的相对不确定度
假定称量的不确定度为 0.2mg,以此计算相对不确定度. *译者注.按照 1g/1000ml 计算溶液制备应为 0.25mg/250ml.

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
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

植物总酚（Total Phenols，TP）试剂盒说明书微量法100T/48S 注意：正式测定之前选择2-3个预期差异大的样本做预测定。

测定意义：植物酚类物质具有清除自由基，抗氧化抗衰老的作用，具有较高的营养价值和医疗保健作用而广泛应用于化妆品、食品、医药等领域。

测定原理：在碱性条件下，酚类物质将钨钼酸还原，产生蓝色化合物，在760nm处有特征吸收峰，测760nm处的吸光值，即可得样品总酚含量。

自备实验用品及仪器：天平、烘箱、粉碎仪、筛子、超声破碎仪、60%乙醇、离心机、可见分光光度计/酶标仪、微量石英比色皿/96孔板、蒸馏水。

试剂组成和配制：提取液：60%乙醇，自备。

试剂一：液体3mL×1瓶，4℃保存。

试剂二：液体5mL×1瓶，4℃保存。

总酚提取：将样本烘干至恒重，粉碎，过40目筛之后，称取约0.1g，加入2.5mL提取液，60℃振荡提取2h。

10000g，25℃，离心10min，取上清，用提取液定容至2.5mL，待测。

测定操作表：1、分光光度计/酶标仪预热30min，调节波长至760nm，蒸馏水调零。

2、操作表对照管测定管样本待测液（µL）10 10试剂一（µL）50混匀，25℃静置2min试剂二（µL）50 50H2O（µL）140 90混匀，25℃静置10 min，于微量石英比色皿/96孔板中，测定760nm吸光值，ΔA=A测定-A 对照。

总酚含量计算公式：a.用微量石英比色皿测定的计算公式如下标准曲线：y = 5.615x+0.0012，R2 = 0.9994总酚含量（mg/g 干重）=（ΔA-0.0012）÷5.615×V样÷（V样÷V样总×W）= 0.445×（ΔA–0.0012）÷WV样总：加入提取液体积，2.5 mL；V反总：反应总体积，0.2mL；V样：反应中样品体积，0.01mL；W：样品质量，g。

FITC快速标记试剂盒FrriendBioio,YourTTrusteddFrienddinScienenticresesearch！AtyouurServvicesassyounneed!5.加⼊适量标标记缓冲液⾄上上述超滤管中，并轻轻吹打混混匀，12,000xg离⼼10min。

并重复操操作多次，直⾄⾄超滤管中没有有蓝⾊，未标记记的FITC被彻底底清除⼲净；收收集超滤管中的溶液，即为FITC标记抗体体。

标记偶联⽐率率的计算:MMWA495/195A495?CF/P3889[A280?(0.35?A4495)]/E0.11%280A2280?(0.35?A495)MMW?E0.1%%280其中C?389?1955C是某个蛋⽩⽩的⼀个常数；MW是蛋⽩的分分⼦量；389是FITC的的分⼦量；195是偶联的的FITC在490nmm（PH13.0）情情况下1mg/ml的吸光值；(0.35XA4955)是FITC在2280nm情况下吸吸光值的校正系系数；E0.1%是1.00mg/ml的蛋⽩⽩在280nm的吸吸光值。

注意事项：1.本试剂盒适适⽤于所有含有有ε-氨基（NNH2-）的⼤分⼦⼦物质的标记，（蛋⽩，抗体体，以及其他含含有伯氨基（NHH2-）的化合物物分⼦），具体标标记⽐例根据待待标记物中氨基基的数量确定；2.本试剂盒中中的FITC助溶溶剂为DMSO，使⽤完毕后要要密封⼲燥保存存；3.本试剂盒配配备了标记物保保存液，实验⼈⼈员也可以根据据被标记物的特点不同，选择更适合的保保存液。

4.本试剂盒未未开封前的有效效期为18个⽉⽉，请在有效期期内使⽤。

5.本系列试剂剂盒提供的为超超滤管分为不同的截留分⼦量量和不同的最⼤容积，客户需要根据⾃⼰⼰要标记的物质的分⼦量⼤⼩⼩和标记量选择择适合的型号；本试剂剂仅供实验室研研究使⽤产品制造商：福因德科技（（武汉）有限公公司FrieendbioScience&Technoology(Wuhann)Co.,Ltd.⽣产地址：武武汉市东湖⾼新新区佳园路光⾕⾕国际B座15层总机机：************技术⽀持：********************⽹址址：?。

蛋白定量试剂使用说明书【产品名称】产品通用名称：蛋白定量试剂产品商用名称:Bradford法蛋白定量试剂盒【包装规格】PP1101（250次）/PP1102（1000次）/PP1103（2500次）【检验原理】Bradford法蛋白浓度定量试剂盒是在世界上常用的蛋白浓度检测方法之一Bradford法基础上（考马斯亮蓝结合显色法）改进而成，当bradford染色液（考马斯亮蓝）和蛋白在酸性条件下结合时，最大吸光值波长立刻由465nm转移至595nm，同时颜色由褐色转为蓝色，通过测定吸光值大小并对照标准蛋白的吸光值，推算出蛋白浓度。

【产品特点】1.灵敏度高，检测浓度下限达到25μg/mL，最小检测蛋白量达到0.5μg，待测样品体积为1-20μL。

2.检测速度极快，10-20个样品只需不足10分钟即可完成。

3.在50-1000μg/mL浓度范围内有较好的线性关系。

【主要组成成分】试剂盒由蛋白标准（5mg/mL BSA），Bradford染色液，使用说明书和合格证组成。

主要组分及储存条件见表1。

表1试剂盒组成、储存试剂盒组成PP1101PP1102PP1103保存蛋白标准（5mg/mL0.25mL1mL 1.5mL×2-20℃BSA）Bradford染色液50mL200mL500mL4℃【储存条件及有效期】1、蛋白标准（5mg/mL BSA）在-20℃条件下保存；Bradford染色液在4℃条件下保存。

2、本产品有效期为一年，请在有效期内使用。

3、为了避免试剂长时间暴露于空气中发生挥发、氧化、pH值变化，各溶液使用后应及时盖紧盖子。

【使用方法】1.取10μL蛋白标准品(5mg/mL BSA)稀释至100μL，使终浓度为0.5mg/mL。

蛋白样品在什么溶液中，标准品也宜用什么溶液稀释。

但是为了简便起见，也可以用0.9%NaCl或PBS稀释标准品。

2.将稀释后标准品（0.5mg/mL BSA）按0,1,2,4,8,12,16,20μL分别加到酶标板中，加标准品稀释液将所有标准品补足到20μL。

8
Function of density and optical thickness
Time
Octet
9
Octet
检测
Baseline Baseline Loading
Octet Biosensors Buffer Ligand-Biotin(ligand) Protein of Interest (analyte) • • • •
15
Small Molecules
0.1 nm
Atoms
Protein - Small Molecule
Octet仪器系统对于科研用户的应用
非标记（直接的）、实时在线、快速地对样品进行测定
与其他分子相互作用检测方法（双向电泳，FP，Co-IP，Western Blot， HPLC，ELISA，NMR，ITC，晶体衍射等 ）互补，BLI技术更快更直接
0
Relative Intensity
Internal Reference (reflection surface 1)
Interface with Liquid (reflection surface 2) Destructive Wavelength interference
6
Interferometric Pattern Changes with Increased Molecular Thickness

OCTET


18

tag GST
Fc,his,FLAG tag KD
biotin
A RNA,DNA
KD =
ቤተ መጻሕፍቲ ባይዱkd ka
R=R0+Req(1-e-kobs*t)

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。

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

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。

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：。

游离胆固醇（FC）含量检测试剂盒说明书微量法注意：本产品试剂有所变动，请注意并严格按照该说明书操作。

货号：BC1895规格：100T/96S产品组成：使用前请认真核对试剂体积与瓶内体积是否一致，有疑问请及时联系索莱宝工作人员。

试剂名称规格保存条件提取液液体100 mL×1瓶（自备）常温保存工作液液体20 mL×1瓶4℃保存标准品粉剂×1支4℃保存溶液的配制：1、提取液：自备异丙醇；2、标准品：10 mg胆固醇，临用前加入517 μL异丙醇，振荡溶解，即为50 μmol/mL的胆固醇标准溶液，再将其用异丙醇稀释为2 μmol/mL的标准品，待测。

产品说明：FC是构成细胞膜的主要成分，也是合成肾上腺皮质激素、性激素、胆汁酸及维生素D等生理活性物质的重要原料。

FC浓度可作为脂代谢的指标。

测定原理：FC氧化酶催化FC生成4-胆甾烯酮和H2O2，过氧化物酶催化H2O2、4-氨基安替比林和酚生成红色醌类化合物，在500nm有吸收峰，其颜色深浅与FC含量成正比。

技术指标：最低检出限：0.119 μmol/mL线性范围：0.125-6 μmol/mL注意：实验之前建议选择2-3个预期差异大的样本做预实验。

如果样本吸光值不在测量范围内建议稀释或者增加样本量进行检测。

需自备的仪器和用品：可见分光光度计/酶标仪、水浴锅、可调式移液器、微量玻璃比色皿/96孔板、研钵/匀浆器、异丙醇、蒸馏水。

操作步骤：一、样本处理（可适当调整待测样本量，具体比例可以参考文献）1.组织：按照组织质量（g）：提取液体积(mL)为1：5~10的比例（建议称取约0.1g组织，加入1mL提取液）进行冰浴匀浆。

8000g，4℃离心10min，取上清置冰上待测。

2.细菌、细胞：按照细胞数量（104个）：提取液体积（mL）为500~1000：1的比例（建议500万细胞加入1mL提取液），冰浴超声波破碎细胞（功率300w，超声2秒，间隔3秒，总时间3min）；然后8000g，4℃，离心10min，取上清置于冰上待测。

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

Save Money by Falling Antibody Clones Earlier
现在我们细胞培养的工作量只有从前的十分之一
“We need to culture only about ten percent of what we used to.” — Director of Biochemistry
的各种筛选工作
“The Octet System gave us the capacity to handle multiple screenings of our library with one instrument in our own lab.” — Screening Lab Manager
ForteBio’s
专利 BLI 技术
Bio-layer Interferometry
Octet System
Label-free，real-time detection of molecular interactions
Octet 蛋白质相互作用工作站
z Label-free detection
无需标记
Octet RED System Specifications
检测技术 传感器 检测结果包括 基于光纤生物传感器的生物膜层光学干涉技术（BLI，BioLayer Interferometry） 根据具体实验，可以使用 10 次

动力学和亲合力分析（kobs，ka，kd，KD） ，结合特异性和结合协同性 Epitope mapping/binning 判断是否有相互作用发生 蛋白质定量 每个样品分析时间：5min-3hr 结合速率常数（ka） ：101-107 M-1s-1 解离速率常数（kd） ：10-6-10-1 s-1 亲合力范围：10-3-10-12M 分子量：>150 da 基线噪音：2.5pm （RMS） 基线漂移：<21pm/hour 分析时间：8 个样品分析时间<2min，96 个样品分析时间<30min 定量范围：0.25ug/ml - 2000ug/ml

octetred96简明实验操作流程Octet Red96是一款高通量生物分析仪器，被广泛应用于蛋白质-蛋白质、蛋白质-配体、蛋白质-细胞之间的相互作用研究。

其操作流程基本相似，下面是Octet Red96简明实验操作流程（动力学）。

1.准备实验样品：根据实验要求，制备并稀释样品，通常是蛋白质溶液。

注意需要留出阴性对照和阳性对照，以及荧光标记的配体或细胞。

2. 运行分析软件：打开Octet Analysis软件，并选择“动力学分析”。

3.设置实验参数：在新建的动力学分析实验页面上，选择运行时间、速度、温度、读取间隔等实验参数。

根据要求选择激光波长和检测模式。

4.图像定点设置：根据阴性对照设置起始基线，以便对后续实验结果进行相对定量分析。

5.样品载板准备：将样品分别加载到不同的96孔板中。

一般使用黑色玻璃底的板，以减少背景信号。

6. 启动实验：将96孔板插入Octet Red96仪器中，点击“Run”按钮开始实验。

仪器首先会进行基线读取，然后开始检测样品与配体/细胞的相互作用。

7.数据分析：实验进行过程中，软件会实时显示传感器偶耦结果。

实验完成后，软件会自动生成动力学曲线，并提供相关数据的定量分析，如关联常数(Kd)、速率常数等。

8.结果解读：根据实验的目的和问题，对实验结果进行解读和分析。

可以根据Kd值来判断样品与配体/细胞之间的亲和性。

9.结果报告：根据实验结果，撰写实验报告或进行数据汇总，附上相应的图表和图像。

10.仪器清洗：实验结束后，将96孔板取出，将仪器内部的传感器进行清洗。

以上是Octet Red96简明实验操作流程（动力学）的详细步骤。

操作前需要充分了解实验原理和仪器操作指南，严格遵守仪器使用规定，并根据具体实验要求进行调整。

实验人员应该具备实验基础知识和技能，以保证实验结果的准确性和可靠性。

Fortebio Octet BLI 数据导出作图步骤
将Octet分析结果的原始数据点导出用于第三方软件作图的操作步骤：●Analysis版本软件：
在数据分析软件的Analysis窗口的左下方Data Export→Export Fitting →选择保存的文件夹→将导出的txt文本复制到excel中。

导出的数据包含了所有浓度的曲线，data为实测的，fit为拟合的，需要将时间处理一下，所有的时间点都扣掉第一个时间点的即可。

●Analysis HT版本软件：
在数据分析软件的Analysis窗口的上方的Export选择Results→选择保存的文件夹→将导出的txt文本复制到excel中。

导出的数据包含了所有浓度的曲线，data为实测的，fit为拟合的，需要将时间处理一下，所有的时间点都扣掉第一个时间点的即可。

（12.2以后的studio直接导出csv文件）。