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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
1
2
3
4
5
6
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
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