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www.nanotemper-technologies.com
Monolith NT.115 SeriesProduct Information
10/2
014
Monolith Instrumentsfor MicroScale Thermophoresis
10/2014
2 www.nanotemper-technologies.com
MicroScale Thermophoresis A technology by NanoTemper
MicroScale Thermophoresis is an easy, fast and precise way to quantify biomolecular interactions. It measures the motion of molecules along microscopic temperature gradients and detects changes in their hydration shell, charge or size.
(QMR\�WKH�EHQH¿WV�RI�067�
2SWLPL]H�DVVD\V�TXLFNO\� ▶ Judge and improve sample quality immediately 0HDVXUH�SUHYLRXVO\�XQPHDVXUDEOH�WDUJHWV� ▶ Work with very small amounts and sensitive samples
%HQH¿W�IURP�FORVH�WR�QDWLYH�FRQGLWLRQV� ▶ Analyze in all buffers and bioliquids (cell lysate, serum) –immobilization-free
'R�\RXU�UHVHDUFK�HI¿FLHQWO\� ▶� (QMR\�SHUIHFW�HDVH�RI�XVH��SXUL¿FDWLRQ�IUHH�PHDVXUHPHQWV� and get rid of maintenance downtime
:RUN�ÀH[LEO\� ▶ Kd s for all molecular weights from ions to ribosomes � � DQG�IRU�S0�WR�P0�ELQGLQJ�DI¿QLWLHV
10/2014
Monolith NT.115 Series | Product Information 3
MicroScale Thermophoresis A powerful technique
MicroScale Thermophoresis (MST) is a powerful technique for quantifying ELRPROHFXODU�LQWHUDFWLRQV��%\�FRPELQLQJ�WKH�SUHFLVLRQ�RI�ÀXRUHVFHQFH�GHWHFWLRQ�ZLWK�WKH�ÀH[LELOLW\�DQG�VHQVLWLYLW\�RI�WKHUPRSKRUHVLV��067�SURYLGHV�D�ÀH[LEOH��UREXVW�DQG�IDVW�ZD\�WR�PHDVXUH�PROHFXODU�LQWHUDFWLRQV��
:KHQ�SHUIRUPLQJ�D�067�H[SHULPHQW��D�PLFURVFRSLF�WHPSHUDWXUH�JUDGLHQW�is induced by an infrared laser, and the directed movement of molecules is GHWHFWHG�DQG�TXDQWL¿HG�XVLQJ�HLWKHU�FRYDOHQWO\�DWWDFKHG�G\HV��ÀXRUHVFHQW�IXVLRQ�SURWHLQV��RU�LQWULQVLF�ÀXRUHVFHQFH�
10/2014
4 www.nanotemper-technologies.com
Discover the Application RangeMicroScale Thermophoresis detects interactions between any kind of biomolecules thus providing a large application range, from ions and small PROHFXOHV�WR�KLJK�PROHFXODU�ZHLJKW�DQG�PXOWL�SURWHLQ�FRPSOH[HV��
Thermophoresis, the movement of molecules in temperature gradients, is not only dependent on the size, but also on the charge and the hydration shell of the molecule of interest. Therefore, binding events can be detected HYHQ�ZLWKRXW�DQ�LQFUHDVH�LQ�VL]H�RU�PDVV�XSRQ�FRPSOH[�IRUPDWLRQ�
Since MST is performed free in solution without any surface immobilization, also bulky or sensitive molecule assemblies such as liposomes, nanodiscs or membrane proteins can be investigated.
MicroScale Thermophoresis (MST)
Bacteria
Cell
Ribosomes
Nucleosomes
Viruses
Liposomes
NanoDiscs
Proteins
Nucleic acids Ions
Nucleotides
Fragments
Atoms
Small Molecules
PeptidesAntibody-FragmentsAntibodies
1000 n
m200
nm
75 nm
7 nm
1 nm
0.1 nm
Size
MW
1000 k
Da
100 kD
a
1 kDa
150 Da
1 Da
10/2014
Monolith NT.115 Series | Product Information 5
Monolith NT.115 and NT.115 Pico
The NT.115 Series measures biomolecular interactions via detection of ÀXRUHVFHQW�G\HV�RU�ÀXRUHVFHQW�IXVLRQ�SURWHLQV��VXFK�DV�*)3��SURYLGLQJ�WKH�IROORZLQJ�EHQH¿WV��
▶ ,PPRELOL]DWLRQ�IUHH�DI¿QLW\�GHWHUPLQDWLRQ�IURP���S0�WR�P0▶ Broad application range▶ %XIIHU�LQGHSHQGHQF\��LQFOXGLQJ�VHUXP�RU�FHOO�O\VDWH▶ 3XUL¿FDWLRQ�IUHH��ÀXRUHVFHQW�IXVLRQ�SURWHLQV
Cat # Instrument Channel 1 Channel 2 $I¿QLW\�5DQJH��Kd) Sample Consumption (per Kd)
*��� NT.115Pico Pico – RED
- 1 pM to mM ����SJ�1
*��� NT.115%OXH�*UHHQ
Nano –BLUE
Nano –*5((1
1 nM to mM ����QJ�2
*��� NT.115 Blue/Red
Nano –BLUE
Nano –RED
1 nM to mM ����QJ�2
*��� NT.115*UHHQ�5HG
Nano –*5((1
Nano –RED
1 nM to mM ����QJ�2
1�FDOFXODWHG�IRU�D�VWDQGDUG�SURWHLQ�RI����N'D�����GDWD�SRLQWV�SHU�Kd DQG����S0�ÀXRUHVFHQWO\�ODEHOHG�SURWHLQ�2�FDOFXODWHG�IRU�D�VWDQGDUG�SURWHLQ�RI����N'D�����GDWD�SRLQWV�SHU�Kd�DQG����Q0�ÀXRUHVFHQWO\�ODEHOHG�SURWHLQ�
10/2014
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Technical Details
Monolith Instruments NT.115Series NT.115 NT.115Pico
Samples per run ���VDPSOHV ���VDPSOHV
)OXRUHVFHQFH�FKDQQHOV�SHU�LQVWUXPHQW 2 �%/8(��5('�RU�*5((1�
1 (RED)
$I¿QLW\�UDQJH 1 nM to mM 1 pM to mM
Labeling required Yes Yes
&RQFHQWUDWLRQ�RI�ÀXRUHVFHQW�PROHFXOH �����������-3 M ��-11�����-3 M
Range of accessible interactions Ŷ�Ŷ�Ŷ�Ŷ�Ŷ Ŷ�Ŷ�Ŷ�Ŷ�Ŷ
Biophysical parameters $I¿QLW\��6WRLFKLRPHWU\��Enthalpy, Enzyme Kinetics
$I¿QLW\��6WRLFKLRPHWU\��Enthalpy, Enzyme Kinetics
7U\SWRSKDQ�ÀXRUHVFHQFH�UHTXLUHG No No
0HDVXUHPHQWV�LQ�FRPSOH[�ELROLTXLGV�(serum, cell lysate)
Yes Yes
Volume per measurement ����ȝO ����ȝO
Molecular weight range (Da) ��1������ ��1������
7LPH�IRU�H[SHULPHQW��DQDO\VLV Minutes Minutes
Immobilization required No No
Temperature controlled 22 - 45 °C 22 - 45 °C
Maintenance required No No
10/2014
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Detectors and Spectra
7KH�H[FLWDWLRQ�HPLVVLRQ�ZDYHOHQJWKV�IRU�WKH�FRUUHVSRQGLQJ�0RQROLWK�NT.115 Series detectors are illustrated below.
7KH�OLJKW�DQG�GDUN�VKDGHG�ER[HV�FRUUHVSRQG�WR�WKH�H[FLWDWLRQ�DQG�HPLVVLRQ�ZDYHOHQJWK�VSHFWUD��UHVSHFWLYHO\��RI�WKH�GLIIHUHQW�ÀXRUHVFHQFH�detectors.
high-throughput MST
96 samples at a time
high versatility: integrate 2 de-tectors to combine high sensitivityfluorescence and label-free applications
integration into liquid-handlingplatform
Monolith NT.Automated
350
[nm]
400
550
450
500
600
650
700
750
300
250
800
LabelFree
NanoBLUE/GREEN
NanoBLUE/RED
NanoGREEN/RED
PicoRed
500
The Monolith instruments harbor different detectors to monitor the fluorescence signalof your sample. The excitation/emission wave-lengths for the corresponding detectors are illustrated below. The Monolith NT.Automatedcan be equipped with a combination of twodifferent detectors.
NT.Automated
96 samples
Yes
combination of up to 2 detectors
Yes
These parameters depend on the
choice of the detectors: The NT.Au-
tomated can be equipped with a
combination of two different de-
tectors which can be freely chosen
from all Monolith detection systems
(Nano, Pico and LabelFree de-
tectors)
< 3 µl
101 - 107
minutes
No
25°C (actively controled)
optional service and performance diagnostic
Monolith Instruments
The white and black shaded boxes correspond to the excitation and emission wavelength spectra of the different fluorescence detectors, respectively.
7
Monolith Detectors
Europe_19052014.qxp 20.05.14 11:03 Seite 9
high-throughput MST
96 samples at a time
high versatility: integrate 2 de-tectors to combine high sensitivityfluorescence and label-free applications
integration into liquid-handlingplatform
Monolith NT.Automated
350
[nm]
400
550
450
500
600
650
700
750
300
250
800
LabelFree
NanoBLUE/GREEN
NanoBLUE/RED
NanoGREEN/RED
PicoRed
500
The Monolith instruments harbor different detectors to monitor the fluorescence signalof your sample. The excitation/emission wave-lengths for the corresponding detectors are illustrated below. The Monolith NT.Automatedcan be equipped with a combination of twodifferent detectors.
NT.Automated
96 samples
Yes
combination of up to 2 detectors
Yes
These parameters depend on the
choice of the detectors: The NT.Au-
tomated can be equipped with a
combination of two different de-
tectors which can be freely chosen
from all Monolith detection systems
(Nano, Pico and LabelFree de-
tectors)
< 3 µl
101 - 107
minutes
No
25°C (actively controled)
optional service and performance diagnostic
Monolith Instruments
The white and black shaded boxes correspond to the excitation and emission wavelength spectra of the different fluorescence detectors, respectively.
7
Monolith Detectors
Europe_19052014.qxp 20.05.14 11:03 Seite 9
high-throughput MST
96 samples at a time
high versatility: integrate 2 de-tectors to combine high sensitivityfluorescence and label-free applications
integration into liquid-handlingplatform
Monolith NT.Automated
350
[nm]
400
550
450
500
600
650
700
750
300
250
800
LabelFree
NanoBLUE/GREEN
NanoBLUE/RED
NanoGREEN/RED
PicoRed
500
The Monolith instruments harbor different detectors to monitor the fluorescence signalof your sample. The excitation/emission wave-lengths for the corresponding detectors are illustrated below. The Monolith NT.Automatedcan be equipped with a combination of twodifferent detectors.
NT.Automated
96 samples
Yes
combination of up to 2 detectors
Yes
These parameters depend on the
choice of the detectors: The NT.Au-
tomated can be equipped with a
combination of two different de-
tectors which can be freely chosen
from all Monolith detection systems
(Nano, Pico and LabelFree de-
tectors)
< 3 µl
101 - 107
minutes
No
25°C (actively controled)
optional service and performance diagnostic
Monolith Instruments
The white and black shaded boxes correspond to the excitation and emission wavelength spectra of the different fluorescence detectors, respectively.
7
Monolith Detectors
Europe_19052014.qxp 20.05.14 11:03 Seite 9
high-throughput MST
96 samples at a time
high versatility: integrate 2 de-tectors to combine high sensitivityfluorescence and label-free applications
integration into liquid-handlingplatform
Monolith NT.Automated
350
[nm]
400
550
450
500
600
650
700
750
300
250
800
LabelFree
NanoBLUE/GREEN
NanoBLUE/RED
NanoGREEN/RED
PicoRed
500
The Monolith instruments harbor different detectors to monitor the fluorescence signalof your sample. The excitation/emission wave-lengths for the corresponding detectors are illustrated below. The Monolith NT.Automatedcan be equipped with a combination of twodifferent detectors.
NT.Automated
96 samples
Yes
combination of up to 2 detectors
Yes
These parameters depend on the
choice of the detectors: The NT.Au-
tomated can be equipped with a
combination of two different de-
tectors which can be freely chosen
from all Monolith detection systems
(Nano, Pico and LabelFree de-
tectors)
< 3 µl
101 - 107
minutes
No
25°C (actively controled)
optional service and performance diagnostic
Monolith Instruments
The white and black shaded boxes correspond to the excitation and emission wavelength spectra of the different fluorescence detectors, respectively.
7
Monolith Detectors
Europe_19052014.qxp 20.05.14 11:03 Seite 9
high-throughput MST
96 samples at a time
high versatility: integrate 2 de-tectors to combine high sensitivityfluorescence and label-free applications
integration into liquid-handlingplatform
Monolith NT.Automated
350
[nm]
400
550
450
500
600
650
700
750
300
250
800
LabelFree
NanoBLUE/GREEN
NanoBLUE/RED
NanoGREEN/RED
PicoRed
500
The Monolith instruments harbor different detectors to monitor the fluorescence signalof your sample. The excitation/emission wave-lengths for the corresponding detectors are illustrated below. The Monolith NT.Automatedcan be equipped with a combination of twodifferent detectors.
NT.Automated
96 samples
Yes
combination of up to 2 detectors
Yes
These parameters depend on the
choice of the detectors: The NT.Au-
tomated can be equipped with a
combination of two different de-
tectors which can be freely chosen
from all Monolith detection systems
(Nano, Pico and LabelFree de-
tectors)
< 3 µl
101 - 107
minutes
No
25°C (actively controled)
optional service and performance diagnostic
Monolith Instruments
The white and black shaded boxes correspond to the excitation and emission wavelength spectra of the different fluorescence detectors, respectively.
7
Monolith Detectors
Europe_19052014.qxp 20.05.14 11:03 Seite 9
10/2014
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7KHUPRSKRUHWLF�DQDO\VLV�RI�D�SURWHLQ�SURWHLQ�LQWHUDFWLRQ��WKLV�LQWHUDFWLRQ�KDV�EHHQ�LQYHVWLJDWHG�LQ�D�SXUL¿HG�V\VWHP�XVLQJ�D�FRYDOHQWO\�DWWDFKHG�ÀXRURSKRUH��$��DQG�XVLQJ�ÀXRUHVFHQW�IXVLRQ�SURWHLQV�LQ�EXIIHU��%��DV�ZHOO�DV�LQ�SXUH�FHOO�O\VDWH��&���
0DWHULDO�ZDV�NLQGO\�SURYLGHG�E\�3URI��*LGHRQ�6FKUHLEHU��:HL]PDQQ�,QVWLWXWH��,VUDHO
-HUDEHN�:LOOHPVHQ��0���$QGUp��7���:DQQHU��5���5RWK��+��0���'XKU��6���%DDVNH��3���DQG�%UHLWVSUHFKHU��'���������0LFUR6FDOH�7KHUPRSKRUHVLV��,QWHUDFWLRQ�DQDO\VLV�DQG�EH\RQG��-RXUQDO�RI�0ROHFXODU�6WUXFWXUH�
10/2014
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5HVXOWV��+LJK�$I¿QLW\�,QWHUDFWLRQV
Detection of picomolar KdV�RI�GLIIHUHQW�H[SHULPHQWDO�V\VWHPV�XVLQJ�WKH�0RQROLWK�NT.115Pico.
Material was kindly provided by Dr. Ute Curth, Medical Unitersity Hanover, and Dr. Ahmed Besheer, Novartis, Basel.
Jerabek-Willemsen, M., André, T., Wanner, R., Roth, H. M., Duhr, S., Baaske, P., and Breitsprecher, D. �������0LFUR6FDOH�7KHUPRSKRUHVLV��,QWHUDFWLRQ�DQDO\VLV�DQG�EH\RQG��-RXUQDO�RI�0ROHFXODU�6WUXFWXUH�
10/2014
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Customer Statements
'U��7LP�6KDUSH��%LRSK\VLFV�)DFLOLW\��%LR]HQWUXP��8QLYHUVLW\�RI� Basel, Switzerland
“We have used our Nanotemper NT.115 MicroScale Thermophoresis (MST) LQVWUXPHQW�H[WHQVLYHO\�WR�VWXG\�PDQ\�GLIIHUHQW�W\SHV�RI�LQWHDFWLRQ��>«@�:KHUH�we have been able to make comparisons, results from MST agree well with WKRVH�IURP�RWKHU�HVWDEOLVKHG�WHFKQLTXHV��,7&��ÀXRUHVFHQFH�LQWHQVLW\�DQG�anisotropy, SPR). However, we particularly appreciate two distinguishing DVSHFWV�RI�067��)LUVWO\��067�FDQ�PHDVXUH�LQWHUSUHWDEOH�VLJQDO�FKDQJHV�LQ�FLUFXPVWDQFHV�ZKHUH�PDQ\�RWKHU�WHFKQLTXHV�VWUXJJOH��L�H��VPDOO�XQODEHOOHG�molecules binding to large labeled molecules, where binding is tight (nM Kd) and one or both partners aggregate at micromolar concentrations. Secondly, RQH�FDQ�RIWHQ�JDLQ�H[WUD�LQIRUPDWLRQ�DERXW�WKH�LQWHUDFWLRQ�SDUWQHUV�IURP�067�titrations, particularly for systems that have changes in their conformational or oligomeric state upon binding. In several cases, multi-phasic titrations KDYH�LQVSLUHG�H[SHULPHQWV�ZLWK�RWKHU�WHFKQLTXHV�WR�FKDUDFWHUL]H�EHKDYLRXUV�WKDW�FDQµW�EH�H[SODLQHG�E\�WKH�VLPSOHVW�ELQGLQJ�PRGHOV�´
Prof. Dr. Uffe Holmskov, Institute for Molecular Medicine, University of Southern Denmark, Odense, Denmark
³:H�DUH�LQWHUHVWHG�LQ�HOXFLGDWLQJ�LQWHUDFWLRQV�LQ�FORVH�WR�QDWLYH�FRQGLWLRQV��thus, we appreciate that the interactions are investigted free in solution and can be studied even in cell lysate. We are using MST for a number of different interactions, mainly to investigate protein-protein interactions but DOVR�WR�VWXG\�SURWHLQ�'1$�LQWHUDFWLRQV�DQG�SURWHLQ�ELQGLQJ�WR�LRQV�´
'U��$OH[H\�5DN��6WUXFWXUDO�%LRORJ\��%LRSK\VLFV��6DQR¿�5'��)UDQFH
³:H�URXWLQHO\�DVVHVV�LQWHUDFWLRQ�DI¿QLW\�IRU�ERWK�VPDOO�PROHFXOH�and biologics projects, with NanoTemper Technologies’ MicroScale Thermophoresis has proved a valuable tool for characterising small molecule-protein and protein-protein interactions, as well as for the study of protein aggregation concentration determination. There is very good agreement with other technologies such as Surface Plasmon Resonance (SPR) and Isothermal Titration Calorimetry (ITC), and we are particularly DSSUHFLDWLYH�RI�WKLV�WHFKQRORJ\�EHFDXVH�RI�WKH�H[WUHPHO\�ORZ�SURWHLQ�FRQVXPSWLRQ�DQG�UHODWLYHO\�VKRUW�WLPH�UHTXLUHG�IRU�WKH�DVVD\�VHWXS�´
10/2014
Monolith NT.115 Series | Product Information 11
Selected Publications
1. 6FKXO]H��5��-���.RPDU��-���%RWWH��0���$OOHQ��:��-���:KLWHKRXVH��6���*ROG��9��$��0���/\FNODPD�D�1LMHKROW��-��$���+XDUG��.���%HUJHU��,���6FKDI¿W]HO��&���DQG�&ROOLQVRQ��,���������0HPEUDQH�SURWHLQ�LQVHUWLRQ�DQG�SURWRQ�PRWLYH�IRUFH�GHSHQGHQW�VHFUHWLRQ�WKURXJK�WKH�EDFWHULDO�KRORWUDQVORFRQ�6HF<(*±6HF')±<DM&±<LG&��3URFHHGLQJV�RI�WKH�1DWLRQDO�$FDGHP\�RI�6FLHQFHV���������������
2. 3DUNHU��-��/���DQG�1HZVWHDG��6���������0ROHFXODU�EDVLV�RI�QLWUDWH�XSWDNH�E\�WKH�SODQW�QLWUDWH�WUDQVSRUWHU�157�����1DWXUH�����������
3. Jerabek-Willemsen, M., André, T., Wanner, R., Roth, H. M., Duhr, S., Baaske, P., and Breitsprecher, '���������0LFUR6FDOH�7KHUPRSKRUHVLV��,QWHUDFWLRQ�DQDO\VLV�DQG�EH\RQG��-RXUQDO�RI�0ROHFXODU�Structure
4. *DIIDURJXOODUL��(��&���.UDXVH��$���%DOER��-���+HUWHQ��'��3���DQG�-lVFKNH��$���������0LFURVFDOH�thermophoresis provides insights into mechanism and thermodynamics of ribozyme catalysis. RNA ELRORJ\���������
5. .KDYUXWVNLL��/���<HK��-���7LPRIHHYD��2���7DUDVRY��6��*���3ULWW��6���6WHIDQLVNR��.���DQG�7DUDVRYD��1���������3URWHLQ�3XUL¿FDWLRQ�IUHH�0HWKRG�RI�%LQGLQJ�$I¿QLW\�'HWHUPLQDWLRQ�E\�0LFURVFDOH�7KHUPRSKRUHVLV��-RXUQDO�RI�9LVXDOL]HG�([SHULPHQWV��H�����
��� (PV�0F&OXQJ��6WHSKDQLH�&���+DLQOLQH��6DUDK�*���'HYDUH��-���=RQJ��+���&DL��6���&DUQHV��6WHSKDQLH�.���6KDZ��6LGQH\�/���DQG�:DOF]DN��&ODLUH�(���������$XURUD�%�,QKLELWV�0&$.�$FWLYLW\�WKURXJK�D�Phosphoconformational Switch that Reduces Microtubule Association. Current Biology
��� Taft, M. H., Behrmann, E., Munske-Weidemann, L.-C., Thiel, C., Raunser, S., and Manstein, D. -���������)XQFWLRQDO�&KDUDFWHUL]DWLRQ�RI�+XPDQ�0\RVLQ���$�DQG�LWV�,QWHUDFWLRQ�ZLWK�)�DFWLQ�DQG�*2/3+���-RXUQDO�RI�%LRORJLFDO�&KHPLVWU\
��� 6FKXO]��6���'ROOHU��$���3HQGLQL��1��5���:LOFH��-��$���3IHLOVFKLIWHU��-���DQG�(EHUKDUGW��:���������'RPDLQ�VSHFL¿F�SKRVSKRPLPHWLF�PXWDWLRQ�DOORZV�GLVVHFWLRQ�RI�GLIIHUHQW�SURWHLQ�NLQDVH�&��3.&��LVRW\SH�triggered activities of the RNA-binding protein HuR. Cellular Signalling
��� $OH[DQGHU��&��*���-�UJHQV��0��&���6KHSKHUG��'��$���)UHXQG��6��0��9���$VKFURIW��$��(���DQG�)HUJXVRQ��1���������7KHUPRG\QDPLF�RULJLQV�RI�SURWHLQ�IROGLQJ��DOORVWHU\��DQG�FDSVLG�IRUPDWLRQ�LQ�WKH�KXPDQ�hepatitis B virus core protein. Proceedings of the National Academy of Sciences
���� ���� 6HLGHO��6��$���:LHQNHQ��&��-���*HLVVOHU��6���-HUDEHN�:LOOHPVHQ��0���'XKU��6���5HLWHU��$���7UDXQHU��'���%UDXQ��'���DQG�%DDVNH��3���������/DEHO�IUHH�PLFURVFDOH�WKHUPRSKRUHVLV�GLVFULPLQDWHV�VLWHV�DQG�DI¿QLW\�RI�SURWHLQ�OLJDQG�ELQGLQJ��$QJHZ�&KHP�,QW�(G�(QJO����������������
11. Xiong, X., Coombs, P. J., Martin, S. R., Liu, J., Xiao, H., McCauley, J. W., Locher, K., Walker, P. A., &ROOLQV��3��-���.DZDRND��<���6NHKHO��-��-���DQG�*DPEOLQ��6��-���������5HFHSWRU�ELQGLQJ�E\�D�IHUUHW�WUDQVPLVVLEOH�+��DYLDQ�LQÀXHQ]D�YLUXV��1DWXUH�������������
12. /LQ��&��&���0HOR��)��$���*KRVK��5���6XHQ��.��0���6WDJJ��/��-���.LUNSDWULFN��-���$UROG��6��7���$KPHG��=���DQG�/DGEXU\��-��(���������,QKLELWLRQ�RI�EDVDO�)*)�UHFHSWRU�VLJQDOLQJ�E\�GLPHULF�*UE���&HOO�����������1524
13. 6HLGHO��6��$���'LMNPDQ��3��0���/HD��:��$���YDQ�GHQ�%RJDDUW��*���-HUDEHN�:LOOHPVHQ��0���/D]LF��$���-RVHSK��-��6���6ULQLYDVDQ��3���%DDVNH��3���6LPHRQRY��$���.DWULWFK��,���0HOR��)��$���/DGEXU\��-��(���6FKUHLEHU��*���:DWWV��$���%UDXQ��'���DQG�'XKU��6���������0LFURVFDOH�WKHUPRSKRUHVLV�TXDQWL¿HV�ELRPROHFXODU�LQWHUDFWLRQV�XQGHU�SUHYLRXVO\�FKDOOHQJLQJ�FRQGLWLRQV��0HWKRGV������������
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10/2
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Europe / International
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