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Biacore
(SPR) Biacore
• Biacore– (Surface Plasmon Resonance, SPR)–– Biacore
• Biacore– SPR– (IFC)–
• Biacore–––
BiacoreTM T200
Biacore
January 2012
3
Biacore
(SPR)
(SPR)
• SPR SPR
•
• 1 RU
1 pg/mm2 ( CM5 )
(SPR)
8
(The Sensorgram)
(Baseline)
(Association)
/ (Steady-state/ Equilibrium)
(Dissociation)
(Baseline)
(Regeneration)
RU
Biacore
• (Yes or No)
• (Specificity)
• -- (Affinity)
• -- (Kinetics)
• (Mechanism)
• (Thermodynamics)
• (Concentration)
mM10-3
μM10-6
nM10-9
• - : 10�14 M
• - : 10�8~10�10 M
• DNA : 10�8~10�10 M
• - 10�6~10-7M
• 10�4~10�10 M
• 10�3~10�6M
(Affinity, KD)
•
•–
SAR–
Carayannopoulos et al (2001) Eur J Immunol, 32, 597-605
• HIV
• HIV
Shuman et al., Antiviral Research 58: 235-42 (2003)
HIV
Biacore
•• DNA/RNA• / / •••• / /
Biacore™ Biacore
•
•
•
•
•
•
•
•
•
…. ….
Biacore
January 2012
16
Biacore
SPR
IFC
(IFC)
Biacore
SPR
IFC
(IFC)
Biacore
SPR
IFC
(IFC)
(IFC)
•••
IFC
( )
(IFC)–
•••
����
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����
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�� ����������
������������
(IFC)–
•••
����
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����
����
�� ����������
������������
(IFC)–
•••
����
����
����
����
�� ����������
������������
50nmCM5
• 2%
•
•
•
•
CM5
11
CM5, CM4, CM3 �
CM7
SA
Biotin CAP
NTA His
L1
HPA
C1
Au
30
GST � GST
NTA His
… …
Biacore
January 2012
28
Analyte
Ligand
Biacore
Biacore
Biacore
Biacore
Biacore
(Immobilization)
Capturing molecule
(Injection)
• (Analyte)
•(response)
•
(Regeneration)
••
�
�
�
Biacore
• SPR
•
•
Biacore
• ( )
• ( )
•
•
Biacore
•
Biacore
Biacore
• Biacore––––––
• Biacore– -– -–
BiacoreTM X100
Biacore
• Biacore
•
Biacore•
•
•
•
•
•
•
– Biacore (IFC) (Auto Sampler)
•
–
–
–
•
•
• = =KD
•• 1:1 50%A B• M•
•–– T II MHC––
�
�
�
•ka
kd
•/
–
KD ka, kd
�
�
�
••
– IgG––
(Eyring plot)
�
�
�
Biacore
HIV
• HIV p6
matrix capsidnuclearcapsid p6
HIV-1 Gag
• Tsg101p6
• Tsg101 (vacuolar protein sorting)
p6 HIV
HIV
anti-GST mAb
Tsg101
HIV-1 p6
GSTGST-HIVp6
Biacore p6/Tsg101
0
400
200
600
0 30 4515Time (s)
Res
pons
e (R
U)
200 �M
67
22
7.5
2.50.80.3
•
•
p6/Tsg101
0
400
200
600
0 30 4515Time (s)
Res
pons
e (R
U)
200 �M
67
22
7.5
2.50.80.3
•
p6/Tsg101
0
400
200
600
0 30 4515Time (s)
Res
pons
e (R
U)
200 �M
67
22
7.5
2.50.80.3
10-6 10-5 10-4 10-3
[Tsg101] (M)10-7
p6/Tsg101
0
400
200
600
0 30 4515Time (s)
Res
pons
e (R
U)
200 �M
67
22
7.5
2.50.80.3
KD = 25 �M
10-6 10-5 10-4 10-3
[Tsg101] (M)10-7
p6/Tsg101
0
20
40
60
80
100
1.E-09 1.E-08 1.E-07 1.E-06 1.E-05 1.E-04 1.E-03 1.E-02
Per
cent
Bou
nd
[Tsg101] (M)
1 5233
2713
KDs = 15 - 27 �M
p6 protein
p6
0
20
40
60
80
100
1.E-09 1.E-08 1.E-07 1.E-06 1.E-05 1.E-04 1.E-03 1.E-02
Per
cent
Bou
nd
[Tsg101] (M)
P10L
P10AP7LA9GT8A
p6
WT20 ��M
Y63A365�M
M95A~1.4mM
Per
cent
Bou
nd
[Tsg101] (M)
Pornillos, O., et al. (2002) EMBO J, 21, 2397.Pornillos, O., et al. (2002) Nat Struct Biol. 9, 812-7.
p6 /Tsg101
• Biacore p6/Tsg101
• p6/Tsg101
• Biacore
• Biacore
p6/TSg101
(ka, kd)
-RNA
Acknowledgements to Dr. Ite Laird-Offringa, USC, LA, USA
Park S. et al, (2000), Moll Cell Biol 20: 4765-72 Katsamba et al, (2001), Biol Chem 276: 21476 -81
• RNA RNA (RBPs)
– RNA
• RNA
- RNA RBP
• Ite Laird-Offringas (USC, Los Angeles) Biacore
RNA
1. RNA
• : 25-125 RU
• : 2M NaCl at 20 μl/min 1 min
-RNA
HuD ( RNA ,RNA RNA
) HuD 3 RNA (RRM1-RRM3)AU RNA
RRM2 RRM3 RRM3HuD RRM1 RRM2
RRM3 RNA
-RNA
• U1A RNA
– U1A: RNA--U1hpII
– U1A RRM, U1hpII RNA
• U1A RNA
–
–
-RNA
20 22 50
56 RNA G4
U1A-U1hpII •
induced fit_
__
__
__
_
U1hpII G-base in hairpin
+
+
+
++
+
U1A
_
__
__
__
_
: “Lure” : “Lock”
Biacore RNA-protein“lure and lock”
G4
Biacore: -RNA
•
• RNA-
• Biacore-
Biacore
Acknowledgements to Avidex LTD
CD80
• Avidex Ltd 5
• T CD80/CD28
( = CD80) - 259 A-E 5- Biacore 259
• on/off --
Data courtesy of Avidex Ltd
•
Data courtesy of Avidex Ltd
•
• CD80 E A-D- Avidex E
• E- PD- E on-rate
E
Data courtesy of Avidex Ltd
Biacore
•
•
•
•
•
•
• KD ( )
•
• ka kd KD
•
-50
0
50
100
150
200
250
300
0 50 100 150 200 250 300 350 400 450
RU
Res
p. D
iff.
sTime
ka kdKD
KD
-15
-10
-5
0
5
10
15
20
25
30
35
40 46 52 58 64 70 76 82 88 94 100 106 112 118 124 130
RU
Res
p. D
iff.
sTime
0
5
10
15
20
25
0 5e-4 1e-3 1.5e-3 2e-3 2.5e-3
Biacore
Biacore
• Biacore•
–––
•••
–––
BiacoreTM 4000
Biacore
Biacore
Biacore
• Biacore
•
• CM5 PI>4• CM4• CM3• C1• CM7• SA• CAP• NTA His-tag• HPA• L1
(Immobilization)
-- /CM5 /
(Immobilization)
Capturing molecule
• CM•••
���� Buffer����
(Pre-concentration)•
+
pH<3.5 3.5<pH<pI
+
-
pH
pH>pI
-
-
pH pH
pH• pH 10mM
•
•
•
RL = Rmax Rmax�100Sm = Analyte:Ligand, Sm=1
=1.5 RL
– RL
Rmax 100 RU ?
Analyte MW = 25,000 Da
Ligand MW = 150,000 DaSm = 1Rmax = 100 RURL = ?
Target
(EDC/NHS)
( )
( )
•�
• (>90%)•
� CM• Buffer
� Tris�
•� IgG
•• His-tag NTA Anti-His•
��
(Bulk Effects)
• (Bulk effect)
•
•
Bulk
Buffer 1
Binding + Bulk
Buffer 1
(Reference surface)•
•
•
Flow cell 1 (Fc1 3)
Flow cell 2 (Fc2 4)
•
• -
• dummy ligand
-- /CM5 /
•
•
•
KD
•
-400
-200
0
200
400
600
800
1000
1200
50 100 150 200 250 300Time s
Resp
onse
RU
• KD• KD• KD
KD
•
•
•
• (Baseline)
•(Binding)
•
Cycle 1 Cycle 2
good
bad
Response
Time
Baseline1
Binding 1
Baseline2
Binding 2
• : •
10%
• : •
• :•
•
•
•:
� pH (10 mM glycine-HCl, pH 3 pH 1.5)� (Ethylene glycol 50%, 75% 100%)� pH (1-50mM NaOH)� MgCl2 (1-4 M)�� Biacore
•?
-- /CM5 /
• 5
•
• KD
•
•
�
��Double Reference
�ka kd KD KD=kd/ka
•
•
•��
•
•
•
• 0.2 μm ( )
• Biacorebuffer
• P20
• ?
• DMSO
• Biacore
•
• ( )
• 20-80% (Rmax)
•
•
•
�
�
� X YKD
•••
••••
Examples- Amine coupling- Ligand thiol coupling- Surface thiol coupling- Maleimide coupling- Aldehyde coupling
Examples- Streptavidin - Biotin- Anti-mouse Ig – MAb- Anti-GST - GST- NTA – 6His- Anti-His – 6His- Anti-FLAG - FLAG
•• SA
• CAP
•DNA
• SAstreptavidin
• DNA
•DNA
•
• pH
•
•
KD
•
•
•
•
•
•
800
800-810-9118
400-810-9118
Biacore
13911728591peiwei.han@ge.com
•––
•– 1 1– KD– ka kd– (MTL)
•––
BiacoreTM 3000
� ? –�
� ? –� – ka� – kd�
•••
• 4 KD = 10 nM = 10-8 M
•
D = d
a
kKk
ka kd(M-1s-1) (s-1)
106 10-2
105 10-3
104 10-4
103 10-5
= 1000 nM
30 min 60 min
Resp
onse
30 min 60 min
= 100 nM
10-2 = 1 minutes10-3 = 12 minutes10-4 = 2 hours10-5 = 19 hours
kd (s-1) t½
•
•
•
3
1:1
A
B
A
BA B
kakd
AB
Association:
Dissociation:
Net rate equation:
whereka
-1[s ]
-1 -1[M s ]kd dissociation rate constant
association rate constant
kd [AB][AB]-ddt
ka[AB]ddt
[A] [B]
ka kd [AB][AB]ddt
[A] [B]
M/s M-1s-1 s-1M M M
The equilibrium constants:
-1[M ]the constantequilibrium association
the dissociation constantequilibrium
kakd
kdka
[AB][A] [B]
[M][AB][A] [B]
At equilibrium:
Association Dissociation
kd [AB][A] [B]ka
s-1M s-1-1 M M M
KA
KD
A Bkakd
AB
ka kd [AB][AB]ddt
[A] [B]
RU/s M RU RU
dRdt ka kdmax R][R RC
AB
has one binding site and reacts with immobilized ligandhas n identical and independent binding sites
s-1M s-1-1
A (analyte)- A
AB -AB R
B (ligand)-B Rmax- B Rmax-R
• KD• KD
Rmax, Req KD
Biacore••
Biacore
•
Mass Transport
•
Analyte ,
• Rmax ( ligand analyte )
• >30ul/min)-- analyte
•
( Rmax )
•
•
• 20-80% (Rmax)
•
•
• (Running buffer)
• 5
•
• KD
•
•
•
•
•
• 1:1
• KD=kd/ka
• Rmax Req
• (MTL)
•
•-50
0
50
100
150
200
250
300
0 50 100 150 200 250 300 350 400 450
RU
Res
p. D
iff.
sTime
ka kdKD
KD
-15
-10
-5
0
5
10
15
20
25
30
35
40 46 52 58 64 70 76 82 88 94 100 106 112 118 124 130
RU
Res
p. D
iff.
sTime
0
5
10
15
20
25
0 5e-4 1e-3 1.5e-3 2e-3 2.5e-3
Biacore
Biacore
13911728591peiwei.han@ge.com
•
•
•
•
•
3
• Biacore!
•
•
4
• (filter) (degas)
-
-
• Prime
- buffer prime
• Standby
5
•- Tools->Working Tools Desorb
SDS/-SDS
• Biacore 3000- connector block injection
port-
•--- Tools ->
Service Tools -> Syringe/Tip
6
•- Tools->Working Tools Desorb
- Tools->Working ToolsSanitize
•- Tools-> Test Tools System
CheckCM5 / /
HBS-EP buffer for Biacore 3000, HBS-N for Biacore T100/T200
7
• 4- Standby
-
• 4- (Shutdown)
70%- Biacore 3000 Connector
block- Biacore X100 T200
Biacore?
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