www.nanotemper-technologies.com

Monolith NT.115 SeriesProduct Information

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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

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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�

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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�

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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

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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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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�

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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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