ì 8ô (SPR) N Ørpb.ioz.cas.cn/syspt/znxz/201401/P020140128523419757443.pdf · 2014-02-07 ·...

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)–

•••

��

��

��

��

��

��

(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

(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

•


•

• CD80 E A-D- Avidex E

• E- PD- E on-rate

E


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

•

•

�

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�ka kd KD KD=kd/ka

•

•

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•

•

• 0.2 μm ( )

• Biacorebuffer

• P20

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• 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

[email protected]

•––

•– 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

[email protected]

•

•

•

•

•

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?

ì 8ô (SPR) N Ørpb.ioz.cas.cn/syspt/znxz/201401/P020140128523419757443.pdf · 2014-02-07 ·...

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Transcript of ì 8ô (SPR) N Ørpb.ioz.cas.cn/syspt/znxz/201401/P020140128523419757443.pdf · 2014-02-07 ·...