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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
Amine Reactivebiosensor
NH2NH2
COOH
Amine Reactivebiosensor
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
KD (M) kon(1/Ms) kdis(1/s) R^2
8.41E-06 1.76E+04 1.48E-01 0.981
KD (M) kon(1/Ms) kdis(1/s) R^2
1.00E-05 1.18E+04 1.18E-01 0.980
KD (M) kon(1/Ms) kdis(1/s) R^2
3.73E-05
6.86E+03 2.56E-01 0.974
KD (M) kon(1/Ms) kdis(1/s) R^2
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
buffer Bio-Ag buffer Ab 1-2 buffer
buffer Bio-Ag buffer Ab 1-4 buffer
buffer Bio-Ag buffer Ab 2-1 buffer
buffer Bio-Ag buffer Ab 2-2 buffer
buffer Bio-Ag buffer Ab 2-3 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
B-capture 2 + MCP-1 - MCP-1 + MCP-1 - MCP-1 + MCP-1 - MCP-1 + MCP-1 - MCP-1
B-capture 3 + MCP-1 - MCP-1 + MCP-1 - MCP-1 + MCP-1 - MCP-1 + MCP-1 - MCP-1
B-capture 4 + 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 1 MCP- 3 Detection 1
B capture 1 MCP- 4 Detection 1
B capture 3 MCP-1 Detection 1
B capture 3 MCP- 2 Detection 1
B capture 3 MCP- 3 Detection 1
B capture 3 MCP- 4 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+
Conc. (nM) KD (M) kon(1/Ms) kon Error kdis(1/s) kdis Error kobs(1/s) Full R^2
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
Conc. (nM) KD (M) kon(1/Ms) kon Error kdis(1/s) kdis Error kobs(1/s) Full R^2
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+
Conc. (nM) KD (M) kon(1/Ms) kon Error kdis(1/s) kdis Error kobs(1/s) Full R^2
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
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Real time
Fluidics-free
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www.fortebio.com