28-9617-44 aa biacore x100 brochure

8/14/2019 28-9617-44 AA Biacore X100 Brochure

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BiacoreGE Healthcare

BiacoreX100The personal Biacore experience


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Boost your protein interactionresearchBiacore X100 is a complete solution for biochemistry, molecular biology,or other research laboratories involved in the study of molecularinteractions. The system contains all the key functionalities needed

for day-to-day molecular interaction research with the purpose ofunderstanding protein function and biological mechanisms.

Biacore systems, used extensively in academic research

worldwide, are cited in almost 10 000 peer-reviewed scientiic

publications.

Real-time monitoring o binding events gives a deep understanding o the

dynamics o molecular interactions, including valuable kinetic data. Biacore

analysis allows or straightorward and rapid data generation, leading to

dependable conclusions and enabling the design o new innovative studies.

Biacore X100 can be used or a broad range o applications, including structure-

unction studies, pathway analysis, drug target identiication and validation,

and assay development.

Versatility and lexibility, in combination with unparalleled support, make it

easy to generate top-quality data or your publication. With Biacore X100 on

your bench, take your experiments to the next level and boost your protein

interaction research.


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-5

0

5

10

15

20

25

30

35

40

-500 0 500 1000 1500 2000 2500 3000

Response(RU)

Time (s)

1 nM

5 nM

10 nM

20 nM

30 nM

Run successul assays rom day one

No regeneration required

Aibody molecules allowed

to partially dissociate rom

immobilized HER2 between

injections (arrows)

Sequential injection o

increasing concentrations

o sample in one

continuous analysis cycle

Biacore X100 uses worklow-orientedsotware that provides a supportive

ramework or assay development

and data interpretation, building your

expertise as you work.

Researchers at Uppsala University used

Biacore X100 to determine the binding

characteristics o various Aibody

molecules to HER2 with regards

to speciicity, ainity, and kinetic

association- and dissociation-rates.

Speciicity was quickly established

by comparing binding levels o

the dierent Aibody molecules to

immobilized HER2 and a reerence

protein. As a next step, the kinetics o

high-ainity binders was studied.

Aibody molecules turned out to

be very diicult to regenerate. This

problem was overcome with single-

cycle kinetics which eliminatesthe need or inding regeneration

conditions.

Acknowledgement:Thuy Tran and Proessor Jrgen

Carlsson, Uppsala University, Sweden

The sotware wizards were easy to use, and the help unction was extremely useul to have.

I got good at this very quickly! Thuy Tran, PhD student, Uppsala University, Sweden

Guided worklows and sotware wizards enable generation o reliable data and rapid development o your expertise.

Single-cycle kinetics sensorgram.


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IdeSIdeS

IdeSIdeS

cystatin

C

+

?

?+

IgG

-1000

0

1000

2000

3000

4000

5000

6000

Response(RU)

0 100 200 300 400 Time (s)

Elucidate complex molecular mechanisms

IdeS is a papain-like cysteineprotease rom the human pathogen

Streptococcus pyogenesthat

speciically degrades IgG. The enzyme

has been studied by Dr. Ulrich von

Pawel-Rammingens group at Ume

University in Sweden. They discovered

that human cystatin C has an

unexpected stimulatory eect on the

IgG-endopeptidase activity o IdeS.

Evidence o an interaction between

the two proteins was irst ound using

a combination o gel iltration and

Western blotting. The mechanisms

o the interactions between IdeS,

cystatin C, and IgG were urther

investigated using Biacore X100.

Their studies showed that cystatin C

binds to IdeS, that IdeS can orm

homodimers, and that IdeS binding

to IgG was not aected by cystatin C.

The irst research paper includingdata rom Biacore X100 was published

only ive months ater the system was

installed.

Having a Biacore X100 instrument in our own lab is o great value or our research and

allows us to employ many new types o studies. Especially the possibility to run SPR

experiments according to our own research agenda is a valuable tool in increasing the

understanding o this sophisticated molecular interaction network.

Dr. Ulrich von Pawel-Rammingen, Ume University, Sweden

Vincents, B. et al. The human proteaseinhibitor cystatin C is an activating coactoror the streptococcal cysteine protease IdeS.Chemistry & Biology15, 960-968 (2008).

Acknowledgement:Dr. Ulrich von Pawel-Rammingen,

Ume University, Sweden

Activity assay or IdeS cleavage o IgG. IgG1binding to

immobilized Protein A is measured. Upper curve shows

a sensogram o uncleaved IgG1, while the lower curve

shows a sensorgram or IgG1cleaved by addition o IdeS.

The precise mechanisms o this complex

network o interactions are now being

urther investigated. To aid in this study,

a standardized enzyme activity assay

or IdeS cleavage o IgG is employed,which is run on Biacore X100.

A schematic diagram showing the

possible interactions between IdeS,

cystatin C, and IgG.

Red lines indicate a stimulatory

eect on the interaction,

illustrated by an arrow.


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0

210-5

410-5

610-5

810-5

110-4

1.210-4

1.410-4

1.610-4

Wild type Cys3Ser/His75Gly

Cys3Ser/Leu73Gly

Cys3Ser/Tyr97Ala

Cys3Ser

Concentration(M)

Concentration from A280

measurement

Concentration from CFCA

ll l l

Wild type Cys3Ser Cys3Ser/His75Gly

Cys3Ser/Tyr97Ala

Cys3Ser/Leu73Gly

105

106

107

ka(M-1s-1)

ka

Wild type Cys3Ser Cys3Ser/His75Gly

Cys3Ser/Tyr97Ala

Cys3Ser/Leu73Gly

ll l l

kd(s-1)

kd

KD

KD(M)

10-4

10-3

10-2

10-1

10-10

10-9

10-8

10-7

Increase the reliability o kinetic analysis

Cystatin B is an inhibitor o papain-like cysteine proteases. Four mutants

were produced in order to study the

importance o three dierent amino

acids at the C-terminal and second

binding loop or its binding to papain.

Since no standard was available,

Calibration-ree concentration analysis

(CFCA) was used to assess protein

concentrations. CFCA is an innovative

tool to measure protein concentrations,

based on speciic binding activity,

without using a standard curve.

The combination o concentration, kinetics, and ainity analysis in a single study enabled

the right interpretation o the interaction mechanism.

Mutants Cys3Ser/His75Gly,Cys3Ser/Tyr97Ala, and Cys3Ser

showed a good agreement between

concentration values obtained

with A280

and CFCA, while mutant

Cys3Ser/Leu73Gly had a very low

concentration as measured by

CFCA. Introduction o mutations

can destabilize protein olding and

prolonged storage or reeze/thaw

procedures may also decrease the

amount o protein capable o binding.

Kinetic analysis based on the A280concentration measurement would

lead to the conclusion that leucine 73

is important or the association rate,

giving about ten times slower binding

than the other mutants. In contrast,

CFCA allowed or correct assessment

o association rate (ka) and ainity (K

D),

allowing an appropriate interpretation

o the interaction mechanism. The

decreased ainities or all mutants

were due to an increased dissociation

rate (kd), while the association rate (k

a)

remained relatively constant.


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-10

-200 300 800 1300 Time (s)

10

30

50

70

90

Response

(RU)H2N

O

O-

-O

O

O

NHHO

N

N

Na+

Na+

O

S

S

O

O

S

O

-200 300 800 1300 Time (s)

Response

(RU)

-5

0

5

10

15

20

25

30

H2N

NH2

S

O O

-200 300 800 1300 Time (s)

Response

(RU)

-5

0

5

10

15

20

25

30

HN

NH2

S

O

OH

ClO

O

O

Develop and run assays involving smallmolecule interactions

Biacore X100 provides the

sensitivity required or challenging

applications. Low molecular weight

(LMW) compounds represent one

challenging application, because

a small molecular size reduces

the signal levels. In addition, LMW

compounds oten require organic

solvents or solubility reasons. The

optional Biacore X100 Plus Package

supports correction o bulk eects

rom high reractive index solvents

such as DMSO.

Human carbonic anhydrases are

known drug targets and inhibitors

are used or treatment o various

diagnoses. The igures show solvent-

corrected sensorgrams o single-

cycle kinetic proiles or urosemide,

azosulamide, and sulanilamide

binding to human carbonicanhydrase II in buer containing 3%

DMSO. Azosulamide has the highest

ainity, due to a slow kdas compared

to the other compounds.

Azosulamide (Mr588) in 3% DMSO binding to

human carbonic anhydrase II.

Sulanilamide (Mr172) in 3% DMSO binding to

human carbonic anhydrase II.

Furosemide (relative molecular mass [Mr] 331),

0.17 to 42.5 M in 3% DMSO binding to human

carbonic anhydrase II.

Add Biacore X100 Plus Package i you are using DMSO or aqueous solubility.


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Antibody selection workflow

Immunization

Testing

Fusion

Selection

Dilution

Lead selection

Animal is immunized with antigen

Test and select the most promisinghybridoma supernatants using

Biacore X100

Biacore X100 is used for selecting thebest monoclonal

Biacore X100 is used to test serum forpresence of specific antibodies

Creation of hybridoma cells

Dilution to create monoclonal colonies

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Make critical decisions with conidence

Mercodia AB develops, manuactures,

and markets in vitrodiagnostic kits

and assays or clinical and research

applications.

Antibody selection is based on

characteristics such as speciicity

andainity. Biacore X100 has

replaced ELISA in Mercodia's

evaluation o monoclonal antibodies

in early product development. This

has resulted in reduced reagent

costs and higher data quality,

leading to signiicantly reduced

project risk.

As Biacore X100 is a critical

component o Mercodias entire

worklow, the reliability o the system

and extensive service support

available rom GE Healthcare were

important aspects in the decision topurchase a Biacore system.

Using Biacore systems allows us to approach our product development and production

questions rom a new angle, providing a better basis or key decision making than we

had previously. Robert Gunnarsson, Product Development Manager, Mercodia AB

Acknowledgement:Robert Gunnarsson, Product

Development Manager, Mercodia AB, Uppsala


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Biacore X100 a complete system solution

Biacore X100 integrates all the components

needed to give quick and reliable molecular

interaction data rom day one. Automated

instrumentation collects high quality data rom

small amounts o sample while minimizing

hands-on time.

The sotware is worklow-oriented and providesa structured, yet lexible ramework or assay

development and data interpretation. A broad

range o consumables are available or various

assay alternatives. Biacore capture kits and

suraces are supported by preprogrammed

worklows in the Biacore X100 sotware and

lexibility is provided through a number o

sotware wizards.

The system sotware helps you build expertise

as you work, with an integrated support

unctionality that provides assay tips andguidelines. In addition, all Biacore X100 users

have access to e-learning tools and an extensive

methodology knowledge database on our Web

site. Biacore X100 provides unparalleled support

rom assay development to data interpretation.

Supporting assay chemistry in capture kits

and sensor suraces allows a broad range o

applications and saves time

Worklow-oriented sotware with built-in guidance ranging

rom assay development to data interpretation

Online tools speed up the learning process


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Robust and accessible

instrumentation or acquisition

o top-quality data


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Get the most out olabel-ree interaction analysis

A comprehensive range o service programs, support tools,

and inormation services is available or Biacore X100.

Instrument serviceDuring installation, dedicated service engineers provide you with quality

service to ensure long-term, trouble-ree perormance o your system.

Service contracts and extended warranty options are available or costcontrol and priority service.

TrainingA range o courses and sel-training tools are available to ensure that you

get the most out o your protein interaction analyses.

Application supportOur experienced application specialists oer customized support to answer

your speciic questions.

Support tools on our Web site

Technical tips and protocols

BIA simulation sotware to perorm dry-run experiments

Interactive tutorials to learn how to setup, run, and evaluate Biacore

analyses

Extensive methodology knowledge database

Material Saety Data Sheets relating to consumables

Download section provides you with the latest sotware version, handbooks,

news, and product-speciic material about your system

Biacore X100 basics e-learning course


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imagination at work

For local ofce contact inormation, visitwww.gelifesciences.com/contact

GE Healthcare Bio-Sciences ABBjrkgatan 30751 84 UppsalaSweden

www.geliesciences.com/biacore

GE, imagination at work, and GE monogram are

trademarks o General Electric Company.

Biacore is a trademark o GE Healthcare companies.

All third party trademarks are the property o their

respective owners.

2009 General Electric Company All rights reserved.

First published Nov. 2009.

GE Healthcare UK Limited, Amersham Place,

Little Chalont, Buckinghamshire, HP7 9NA, UK

GE Healthcare Europe, GmbH, Munzinger Strasse 5,

D-79111, Freiburg, Germany

GE Healthcare Bio-Sciences Corp.

800 Centennial Avenue, P.O. Box 1327

Piscataway, NJ 08855-1327, USA

GE Healthcare Japan Corporation

Sanken Bldg., 3-25-1, Hyakunincho

Shinjuku-ku, Tokyo 169-0073, Japan

28-9617-44 AA 11/2009

28-9617-44 aa biacore x100 brochure

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