Scott Zhou, North China FAS MB:15810470035, Email:[email protected] Mar.20 th, 2013 Octet...

Scott Zhou, North China FASMB:15810470035, Email:[email protected]

Mar.20th, 2013

Octet TrainingPart II: Kinetics on the Octet

Agenda

Basic Kinetics

BLI Kinetics Workflow

BLI Kinetics Applications

Basic Kinetics

Distance between the two reflecting

surfaces = ℓ

Intensity λ =ƒ(λ, ℓ)R

ela

tiv

e I

nte

ns

ity

Wavelength (nm)

100%

0

nm

sh

ift

Time

可实时检测到两个反射表面间距的改变

Biolayer Interferometry （ BLI ）

Introduction to Basic Kinetics

• Definitions of ka, kd, KD, and kobs

• Calculation of ka, kd, KD, and kobs

• Concentration Dependent vs. Concentration Independent Parameters

Y=Y0+Ae-kd*t Y=Y0+A(1-e-kobs*t)

kobs – kd

[Conc Ag (M)]ka=

Ab + Ag Complexka

kd

KD = kd

ka

Our analysis follows a 1:1 binding model:

ka = rate of association or “on-rate”; unit = M-1sec-1

kd = rate of disassociation or “off-rate”; unit = sec-1

Non-linear curve fit of data

Binding Kinetics: Overview

,unit = M

• Note that ka (or kon) and KD (affinity value) are concentration dependent (M or molar in the units). Kd (or koff) is a concentration independent value. Kd can be used to screen crude samples relative to each other.

Real-Time Kinetics Provides Better Binding InformationOver ELISA Techniques

• ELISA methods only provide approximations of KD.

• Different combinations of Kon and Koff can give the same affinity measurement.

• These two compounds appear to have the same KD, however Example B has a 100x tighter dissociation than Example A. For this pharmaceutical application, a tighter dissociation would be preferred.

• Washing steps in ELISA methods will remove weak antibody interactions, and thus are not feasible to use for characterization.

Association Dissociation KD = Affinty

Example A 1.00E+04 1.00E-02 1.00E-06

Example B 1.00E+02 1.00E-04 1.00E-06

-0.5

0.5

1.5

2.5

3.5

4.5

5.5

0 100 200 300 400 500 600 700

Time (sec)

nm

sh

ift

example A

example B

BLI Kinetics Workflow

Getting Started

• Computer & Instrument start-up• Sensor handling• Loading of the instrument

Getting Started

• Turning On the system:1. Turn on the computer. 2. Next, make sure the sensor and sample positions are clear inside the

Octet and turn on the power supply. 3. 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.

• Setting up experiment• Use 200 µL sample in each well.• Pre-wet the sensors in buffer/media (time may vary depending on sensor

– consult product insert).

Pre-wetting The Sensors

Be sure to get the corner of the 96 well plate under the lip of the sensor tray. If the 96 well plate is above the lip, the sensor tray will be out of alignment and the sensors will not be picked up by the optics box properly.

Keep the sensors in the pre-wet plate at all times when running an assay.

Correct Incorrect

Inserting the Sensor Tray into the Sensor Tray Holder and the Sample Plate into the Sample Plate Holder

1-The sensor tray is “keyed” to fit into the holder in only one way (it looks vaguely like an arrow and it points into the instrument). 2-The Sample Plate holder is marked “A1” (Red Circle) which corresponds with well “A-1” of the 96 well plate.3-Place the sample plate into the sample plate holder4-Proper placement of the sensor tray and sample plate in the Octet.

1 2 3

4

Biosensor selectionAccording to application & sample type etc.

新开发了四种传感器1. Anti-GST Biosensor: 用于含 GST 标签的蛋白2. NTA Biosensor ：用于含 His 标签的蛋白3. Anti-human Fab-CH1 ：用于人 Fab, F(ab’)2 及 Ab1~44. Anti-Flag biosensor ：用于含 Flag 标签的蛋白

Ligand immobilizationAccording to application & sample type etc.

Minimal biotinylation

(in solution)

Biotin–linker-NHS

NH2

H2N

NH2

Biotin

H2N

Biotin

NH2NH2

Immobilization

Streptavidin biosensor

Streptavidinbiosensor

SA, SSA 传感器的偶联：中性环境

AR2G 传感器的偶联：酸性环境， pH 条件一般需优化

（类）共价键偶联方式：结合稳定，无偶联特异性；要求偶联分子纯度高，浓度要求 ug/ml 级别；偶联的缓冲液不能含有氨基组分（如 Tris ），不能含有载体蛋白如BSA 等（如有则需先替换），特别适合高亲和力检测。

Activation

EDC/NHS

Immobilization

NH2

H2NCOOHCOOH COOH

Amine Reactivebiosensor

NHSNHS NHS


NH2NH2

COOH


Capture protein

Analyze kinetics

AHC, AMC ， Anti-GST, Ni-NTA 等传感器的偶联

（类）亲和偶联方式：偶联特异性高，偶联结合力较共价键弱；对靶标样品纯度要求不高，粗样可直接用来偶联（如含靶标分子的培养液，裂解液，腹水等）；高亲和力相互作用推荐选择（类）共价键偶联方式。

Ligand immobilizationAccording to application & sample type etc.

AssociationSample & concentration setting-important

• Pre-test 1:1. Relative high concentration(10*KD/100*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. 3. Blank control.4. Data quality determined by R/X square in addition to compare with other

source data.

BufferLigand-BiotinProtein of Interest Bi

ndin

g (n

m)

Time

BaselineLoading

Baseline

Association Dissociation

• 8 or 16 samples can be analyzed in parallel• Measure on rates and off rates• Data is displayed in real-time• Experimental protocols can be customized

Octet Biosensors

Kinetics workflow

Biosensor regenerationDepends on ligand & interaction characteristics etc

SA, SSA,AR 的再生

AHC, AMC,Anti-GST, Anti-His 的再生

1. SA,SSA,AR 等传感器偶联靶标时，主要通过共价连接的方式，结合力强，再生条件不会将靶标洗脱下来，无需重新偶联（ Loading ）。

2. AHC 等传感器主要通过特异的亲和将靶标分子偶联至传感器，结合力较共价键弱，再生条件下，靶标将会被洗脱下来，需重新偶联(Loading) 。

• 再生步骤：在仪器中在线完成，只需在微孔板中额外加一列再生缓冲液 buffer 。

• 传感器的再生条件：强酸，强碱，高盐缓冲液。

SA, SSA, AR 传感器的再生

Regenerate

Target protein removed

Capture protein

Analyze kinetics

Binding of biotinylated receptor

Association/ dissociation of target protein

Original streptavidin sensor surface

• 再生效果跟偶联蛋白的稳定性有很大关系。• 再生条件可根据传感器使用说明推荐的条件进行，也可实验室根

据自身样品自行优化再生条件。

AHC, AMC ， Anti-His, Anti-GST 传感器的再生

Regenerate

Sensor back to original surface

Capture protein

Analyze kinetics

Loading with IgG or Fc containing ligand

Association and dissociation of target protein

Original sensor surface

• 再生之后需重新 Loading ，之后再进行动力学检测。• 与 SA 和 AR 不同，这类传感器再生之后，可偶联不同的靶标。• 对偶联蛋白本身的稳定性要求不高。

NTA 传感器的再生

Regenerate

Target and NiCl2 removed

Capture HIS-tagged protein

Analyze kinetics

Functional surface target captured

Association/ dissociation or quantitation of analyte protein

Original Nickel NTA sensor surface

• 与镍柱再生类似。• 再生之后，较于 AHC, AMC, Anti-GST 等不同的是：需要对传感

器 Recharge (NiCl2)

Recharge with NiCl2

BLI Kinetics Applications

小分子化合物筛选、亲和力测定

Initial Screening of a Focused Library336 compounds screened and processed in one 384 well plate

Blue = target sensorRed = Control sensor

Small molecule screening-Novartis

Hit confirmation

kon: 5E4 M-1s-1

koff: 7E-2 s-1

KD: 1.4 uM

S

S

OO

NH2

ClCl

O

O NH2

SOO

NH2

Cl

Cl

NN

O

CH3

S

O O

NH2

Cl

F

CH3

NN

O

CH3CH3

CH3

CH3

CH3

Buffer

Negative control

Positive control

O

OH

OHN

NH2

N

O

OH

N

NH2

Non-binder

Non-binder Non-binder Aggregator

Aggregator kon: 2E5 M-1s-1

koff: 1E-1 s-1

KD: 613.5 nM

kon: 9E2 M-1s-1

koff: 1E-1 s-1

KD: 140.4 uM

kon: 2E5 M-1s-1

koff: 1E-1 s-1

KD: 597.8 nM

kon: 3E3 M-1s-1

koff: 8E-2 s-1

KD: 24.4 uM

kon: 8E5 M-1s-1

koff: 6E-2 s-1

KD: 73.9 nM

Steady-state Analysis of Confirmed Hits

S

O O

NH2

Cl

F

CH3

Rack7-H3Steady state KD: 66 nMKinetic KD: 74 nM

Rack3-H5Steady state KD: 26 µMKinetic KD: 24 µM

NN

O

CH3

Rack1-F5Steady state KD: 560 nMKinetic KD: 598 nM

SOO

NH2

Cl

Cl

O

O NH2

Rack 1-D10Steady state KD: 130 µMKinetic KD: 140.4 µM

Rack1-C6Steady state KD: 630 nMKinetic KD: 613.5 nM

S

S

OO

NH2

ClCl

FurosemideSteady state KD: 1.3 µMKinetic KD: 1.4 µM

Steady-state analysis is in agreement with Kinetic analysis

Furosemide （呋喃苯氨酸） (MW 330D)

Acetazolimide 乙酰唑胺(MW 222D)

Sulpiride （硫苯酰胺） (MW 341D)

Each compound run in a titration series of 5 concentrations in duplicate.

All data from one walk away run

Small molecule-Protein interaction

KD (M) kon(1/Ms) kdis(1/s) R^2

2.34E-04

3.09E+02 7.22E-02 0.913


8.41E-06 1.76E+04 1.48E-01 0.981


1.00E-05 1.18E+04 1.18E-01 0.980


3.73E-05

6.86E+03 2.56E-01 0.974


1.04E-04

1.59E+03 1.65E-01 0.985

KD (M) kon(1/Ms)kdis(1/s)

R^2

8.19E-06

7.84E+036.42E-

020.976

KD (M)kon(1/Ms)

kdis(1/s) R^2

3.62E-06

2.80E+04 1.01E-01 0.980

Small molecule-Protein interaction

Sensor type: SSABuffer: PBS + 1% DMSO 数据来自 Beigene Inc.

蛋白 - 蛋白、蛋白 -DNA 、蛋白 - 糖分子

Kinetics of Arabidopsis Proteins measured on Octet

WT Mutant Despite gross changes in plant growth rate, Octet data demonstrates the kinetic parameters of the mutated kinase are unchanged from wild type.

Octet QK parameters:

• Standard 96W plate• Anti-Murine biosensors• 30C, 1000 RPM in MOPS buffer• Loaded anti-GST antibody to

capture GST-BAK protein• 15 minute association w/ BRI1• 15 minute dissociation

DNA-Binding Protein Kinetic Analysis on the Octet QK

Binding of DNA-Binding Protein to Immobilized Biotinylated ssDNA

Rapid Analysis of Binding of Transcription Factors and Other Promoter Elements to Specific DNA Sequences

kd ka KD

4.78E-055.59E+0

4 8.55E-10

Biotin-DNA

Protein Dissociation in Buffer

Saccharide-Protein

Data from SWU from Glyconex,Taiwan

克隆筛选、抗体筛选、抗体 - 抗原亲和力测定、抗体配对

Antibody Clone selection Using the Anti-Murine Biosensor to Rank Order Clones

Binding of 7 antibody supernatants in DMEM media supplemented with 15%

horse serum

Able to quickly bin into high, medium and low producing cell lines

The Octet can also be used to monitor the purification process in addition to

screening of clones

Assay took less than 20 minutes to set up and run on Octet QK

High

Medium

Low

Blank Media

Antibody Screening

Baseline Loading Baseline association dissociation

buffer Bio-Ag buffer Ab 2B buffer

buffer Bio-Ag buffer Ab 2D buffer

buffer Bio-Ag buffer Ab 1-1 buffer






Sensor type ： SASample ： supernatant without dilution.数据来自军事医学科学院 .

Ab Screening & Order rank results

Sample IDResponse kdis(1/s)

kdis Error

mAb 2B 0.4546 6.23E-03 1.41E-04mAb 2D 0.4178 5.68E-03 1.61E-04mono Ab 1-1 0.7806 6.19E-03 1.42E-04mono Ab 1-2 1.0178 1.08E-02 2.39E-04mono Ab 1-4 1.0235 7.25E-03 1.64E-04mono Ab 2-1 0.4227 4.78E-03 3.03E-04mono Ab 2-2 0.8906 5.24E-03 1.53E-04mono Ab 2-3 0.993 6.23E-03 1.36E-04

数据来自军事医学科学院 .

Detection of anti-influenza virus antibodies in diluted serum to a his-tagged HA antigen immobilized on Anti-

Penta His Biosensors on the Octet RED

Ab-Ag interaction Kinetic analysis of anti-influenza antibodies using a His-tagged Antigen

Carney, PJ et al. CLINICAL AND VACCINE IMMUNOLOGY, Sept. 2010, Vol. 17, No. 9

Screen for Antibody Specificity and AffinityYes / No Binding Can be Visualized Quickly

detection 1 detection 2 detection 3 detection 4

B-capture 1 + MCP-1 - MCP-1 + MCP-1 - MCP-1 + MCP-1 - MCP-1 + MCP-1 - MCP-1




Buffer

B-capture 1

B-capture 2

B-capture 3

B-capture 4

MCP-1 (analyte)

Detection 1

Detection 2

Detection 3

Detection 4

A small diagnostic company needed a method to screen commercial Ab sandwich pairs against identified biomarkers to develop a bead-based diagnostic platform.

Plate layout was set up to screen 4 capture antibodies against 4 detection antibodies. 32 combinations were tested in 1 hour.

Data Analysis Tools Can Quickly Identify Successful Pairs

DetectionAb 1

Detection Ab2

Detection Ab3

Detection Ab4

Capture Ab1 + - - -

Capture Ab2 + - - +

Capture Ab3 + - - +

Capture Ab4 - - - -

16 antibody pairs screened, with and without MCP-1.All 5 pairs confirmed specificity and did not bind without analyte.

B capture 1 MCP-1 Detection 1

B capture 1 MCP- 2 Detection 1



B capture 3 MCP-1 Detection 1




Both selected pairs are specific for MCP-1 and do not bind MCP-2, MCP-3 or MCP-4

抗体 / 蛋白 - 病毒亲和力测定蛋白 - 细菌亲和力测定蛋白 - 纳米颗粒亲和力测定蛋白聚合 ( 蛋白 - 纳米颗粒 )蛋白 -RNA 亲和力测定膜蛋白亲和力测定（蛋白 - 蛋白）定量测定（粗制样品）中药研究（蛋白 - 多糖）

Cases Octet more preferable

Antibody-Virus

virus1

virus2

virus3

Neg. virus

Anti-virus antibody

数据来自中科院生物物理所。

steps: baseline-loading(bio-pro)-baseline-asso(E.Coli)-diss.(PBS)

Protein-bacteria interaction

Sensor type: SABuffer: PB + 0.5%BSA + 0.2% tween-20数据来自中国农业大学。

Protein- Q-dot

Data from Wuhan institute of virology,CAS

Immobilize protein on SA sensor surface and dilute quantum dot in solution as analyte

Aggregation :protein-nanoparticle interaction

Object: bio-pro vs nanoparticleSolution : Loading bio-pro + nanoparticle

Background: proteins form fibers in neutral solution, while in acidic solution the nanoparticle promotes fiber formation, both of which couldn’t be detected with ITC or SPR.

Buffer: pH3.0 HAc;

Outcome: good data.

Aggregation :protein-nanoparticle interaction

Sample ID KD (M) kon(1/Ms) kon Error kdis(1/s) kdis Error kobs(1/s) Full R^2P1 2.95E-09 8.07E+04 5.91E+02 2.38E-04 8.08E-06 4.06E-02 0.993833P1 2.95E-09 8.07E+04 5.91E+02 2.38E-04 8.08E-06 2.04E-02 0.993833P1 2.95E-09 8.07E+04 5.91E+02 2.38E-04 8.08E-06 1.03E-02 0.993833P1 2.95E-09 8.07E+04 5.91E+02 2.38E-04 8.08E-06 5.28E-03 0.993833P1 2.95E-09 8.07E+04 5.91E+02 2.38E-04 8.08E-06 2.76E-03 0.993833P1 2.95E-09 8.07E+04 5.91E+02 2.38E-04 8.08E-06 1.50E-03 0.993833P2 1.22E-08 1.39E+04 1.06E+02 1.71E-04 8.11E-06 1.41E-02 0.996337P2 1.22E-08 1.39E+04 1.06E+02 1.71E-04 8.11E-06 7.14E-03 0.996337P2 1.22E-08 1.39E+04 1.06E+02 1.71E-04 8.11E-06 3.66E-03 0.996337P2 1.22E-08 1.39E+04 1.06E+02 1.71E-04 8.11E-06 1.91E-03 0.996337P2 1.22E-08 1.39E+04 1.06E+02 1.71E-04 8.11E-06 1.04E-03 0.996337P2 1.22E-08 1.39E+04 1.06E+02 1.71E-04 8.11E-06 6.06E-04 0.996337

Pro-P1 Pro-P2

数据来自中科院高能所。

KD=6.22E-07,R2=0.988

RNA-ProteinRNA immobilized to biosensors.

Bio-RNA loading(10nM)

10uM

5uM

2.5uM

1.25uM

0.625uM

• No NSB signal between 10uM protein and blank sensor without bio-RNA(data not showed) • Actually 5min is enough for association step• Compare with BIACORE, no need to regenerate sensors immobilized RNA.• 数据来自于中科院上海生化与细胞所。

Associated with protein

Protein-RNAProtein immobilized to biosensors.

Conc. (nM) KD (M) kon(1/Ms) kon Error kdis(1/s) kdis Error kobs(1/s) Full R^2200 2.97E-09 5.21E+05 4.41E+03 1.55E-03 9.22E-06 1.06E-01 0.989572100 2.97E-09 5.21E+05 4.41E+03 1.55E-03 9.22E-06 5.37E-02 0.98957250 2.97E-09 5.21E+05 4.41E+03 1.55E-03 9.22E-06 2.76E-02 0.98957225 2.97E-09 5.21E+05 4.41E+03 1.55E-03 9.22E-06 1.46E-02 0.989572

12.5 2.97E-09 5.21E+05 4.41E+03 1.55E-03 9.22E-06 8.06E-03 0.989572

数据来自军事医学科学院。

Membrane protein-protein interaction

Object: pro vs proSolution : Loading bio-pro + pro

Background: there is high percentage of detergents in membrane protein samples thus could not be measured on SPR-based system due to bubbles generated by the buffer.

Buffer: assay buffer containing detergents from customer;

Outcome: good data.

Conc. (nM) KD (M) kon(1/Ms) kon Error kdis(1/s) kdis Error kobs(1/s) Full R^2

2.5 2.14E-08 2.75E+06 8.04E+04 5.89E-02 1.32E-03 6.58E-02 0.934619

5 2.14E-08 2.75E+06 8.04E+04 5.89E-02 1.32E-03 7.26E-02 0.934619

10 2.14E-08 2.75E+06 8.04E+04 5.89E-02 1.32E-03 8.64E-02 0.934619

20 2.14E-08 2.75E+06 8.04E+04 5.89E-02 1.32E-03 1.14E-01 0.934619

40 2.14E-08 2.75E+06 8.04E+04 5.89E-02 1.32E-03 1.69E-01 0.934619

80 2.14E-08 2.75E+06 8.04E+04 5.89E-02 1.32E-03 2.79E-01 0.934619

0 Ca2+ 100nM Ca2+


2.5 1.61E-08 3.66E+06 8.60E+04 5.88E-02 1.07E-03 6.79E-02 0.959003

5 1.61E-08 3.66E+06 8.60E+04 5.88E-02 1.07E-03 7.71E-02 0.959003

10 1.61E-08 3.66E+06 8.60E+04 5.88E-02 1.07E-03 9.54E-02 0.959003

20 1.61E-08 3.66E+06 8.60E+04 5.88E-02 1.07E-03 1.32E-01 0.959003

40 1.61E-08 3.66E+06 8.60E+04 5.88E-02 1.07E-03 2.05E-01 0.959003

80 1.61E-08 3.66E+06 8.60E+04 5.88E-02 1.07E-03 3.52E-01 0.959003


2.5 1.73E-08 2.84E+06 6.28E+04 4.93E-02 8.14E-04 5.64E-02 0.959743

5 1.73E-08 2.84E+06 6.28E+04 4.93E-02 8.14E-04 6.35E-02 0.959743

10 1.73E-08 2.84E+06 6.28E+04 4.93E-02 8.14E-04 7.77E-02 0.959743

20 1.73E-08 2.84E+06 6.28E+04 4.93E-02 8.14E-04 1.06E-01 0.959743

40 1.73E-08 2.84E+06 6.28E+04 4.93E-02 8.14E-04 1.63E-01 0.959743

80 1.73E-08 2.84E+06 6.28E+04 4.93E-02 8.14E-04 2.77E-01 0.959743

100uM Ca2+

Membrane protein-protein interaction

数据来自北大医学部。

1mM Ca2+


1.25 7.59E-09 3.33E+06 1.04E+05 2.53E-02 4.72E-04 2.94E-02 0.893988

2.5 7.59E-09 3.33E+06 1.04E+05 2.53E-02 4.72E-04 3.36E-02 0.893988

5 7.59E-09 3.33E+06 1.04E+05 2.53E-02 4.72E-04 4.19E-02 0.893988

10 7.59E-09 3.33E+06 1.04E+05 2.53E-02 4.72E-04 5.85E-02 0.893988

20 7.59E-09 3.33E+06 1.04E+05 2.53E-02 4.72E-04 9.18E-02 0.893988

研究背景 :

• 中药成分复杂，光吸收法、荧光法等无法有效确证其细胞及活体水平研究结果；

• 利用传统 SPR 技术在原理上检测依赖于折光率变化，但很多碳水化合物与 buffer 折光率一致，不易引起折光率明显变化；进样模式上依赖于微流控系统，成分复杂容易造成系统堵塞；

• BLI 检测采用非标记技术、在微孔板中检测速度更快、通量更高，为复杂样品的确证提供有力工具。

中药药理研究中的应用

蛋白 X 与多糖相互作用

目的 :

筛选蛋白 X （含 Fc片段）与不同多糖相互作用，确定其动力学参数；

样品信息：多糖：（包括阳性对照） 12 种，均由聚合度不同糖链构成，分子量未知，筛选浓度为 1mg/ml 及 2mg/ml ；

靶标：蛋白 X （含 Fc段），总量 50ug ，以 PBS配制成 25ug/ml溶液；

传感器： AHC sensor.

Buffer ： PBS

Positive binding

Pos. Contr.: Positive binding?

结果：从 12 种多糖中初步筛选中发现 3 种可与蛋白 X 结合，阳性对照结合曲线可能因所用浓度过高所致。

将蛋白 X 固相化到 AHC 传感器上，检测浓度为 1mg/ml 及 2mg/ml 多糖，结合信号为阳性样品进一步采用浓度梯度确证 .

蛋白 X 与多糖相互作用

Bio-Pro X ： 25ug/ml LPS ： 15ug/ml~1.25ug/ml

成分复杂，摩尔浓度未知，仅拟合解离常数， Kd=0.255/s ；

根据曲线快上快下特征，初步特测阳性对照中与蛋白 X 相互作用成分可能为低分子量物质，如小分子等。

阳性对照动力学检测

Bio-Pro X ： 25ug/ml 多糖 A ： 0.5mg/ml~0.03mg/ml

成分复杂，摩尔浓度未知，假设分子量为 180kDa ，拟合所得KD=4.09nM ；

根据曲线特征及信号大小，初步推测多糖 A 中与蛋白 X 相互作用成不会是低分子量物质。

多糖动力学检测

Summary

Workflow

• Select biosensors

• Pre-wet sensors

• Prepare samples

• Pre-test 1(Yes/No binding, High concentration-10/100 fold KD, Pos.cont. Screening)

• Pre-test 2(determine concentration dilution range)

• KD test(no less than 4 conc.)

• KD calculation

Keys & Optimization

• Set proper control(pos. & neg. control)

• Know KD from publications or other experiment and etc.

• Assay buffer optimization(BSA & Tween-20)

• Change sensor type

Pall ForteBio 解决方案

Label-free

Real time

Fluidics-free

Fast ， Accurate ， Easy.

www.fortebio.com

http://www.fortebio.com/

Scott Zhou, North China FAS MB:15810470035, Email:[email protected] Mar.20 th, 2013 Octet...

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Transcript of Scott Zhou, North China FAS MB:15810470035, Email:[email protected] Mar.20 th, 2013 Octet...