Nano Temper Monolith NT.115 User manual

User Manual
Monolith®NT.115

2
Table of Contents
Safety Considerations.....................................................................................................................3
Regulatory Statement.....................................................................................................................6
Specifications..................................................................................................................................7
Preface...........................................................................................................................................9
Notices............................................................................................................................................9
Limited Warranty...........................................................................................................................10
Installation Requirements..............................................................................................................11
Installation and Connecting Cables...............................................................................................12
Maintenance and Operation..........................................................................................................13
Waste Treatment ..........................................................................................................................13
1. Introduction to MicroScale Thermophoresis (MST)...................................................................14
1.1. Method........................................................................................................................14
1.2. Literature.....................................................................................................................17
2. Monolith®NT.115 Instrument....................................................................................................18
2.1. Models and Most Common Fluorophores.................................................................... 18
2.2. Application Range.......................................................................................................18
2.3. Sensitivity....................................................................................................................18
2.4. Sample Consumption..................................................................................................18
2.5. Capillary Format.......................................................................................................... 19
2.6. Close-to-Native Conditions..........................................................................................19
2.7. Dedicated MO.Control Software and MO.Affinity Analysis Software............................19
3. Preparation of MST Experiments..............................................................................................20
3.1. Control Kits - Performing Standard Experiments .........................................................20
3.2. Selecting the Right Monolith Capillary.........................................................................20
3.3. Fluorescence Labeling ................................................................................................21
3.4. Sample Loading.......................................................................................................... 21
4. Using the Monolith®NT.115 Instrument....................................................................................23

3
Safety Considerations
To ensure operation safety, this instrument must be operated correctly and maintained according to
a regular schedule. Carefully read to fully understand all safety precautions in this manual before
operating the instrument. Please take a moment to understand what the signal words WARNING!,
CAUTION and NOTE mean in this manual.
Safety symbols
WARNING!
AWARNING! indicates a potentially hazardous situation
which, if not avoided, could result in serious injury or even
death.
CAUTION
ACAUTION indicates a potentially hazardous situation which,
if not avoided, may result in minor or moderate injury. It may
also be used to alert against damaging the equipment or the
instrument.
Do not proceed beyond a WARNING! or CAUTION notice until
you understand the hazardous conditions and have taken the
appropriate steps.
NOTE
ANOTE provides additional information to help the operator
achieve optimal instrument and assay performance.
Read manual label. This label indicates that you have to read
the manual before using the instrument. This label is
positioned at the back of the device.
Warning symbol. This symbol indicates laser radiation, it is put
on products which have a laser built in. This warning label is
positioned at the back of the device.
Identification label. This label is
positioned at the back/ rear panel of the
device.

4
WARNING! Only operate the Monolith NT.115 instrument with the supplied external power supply
(SPU63-105, Sinpro Electronic Co Ltd or Ael60US12, XP Power LLC or GS90A12-P1M, MeanWell
or GS60A12-P1J, MeanWell). Onlyuse the supplied cables andplugs. If not doing so yourisk electric
shock and fire.
WARNING! Do not operate the Monolith NT.115 with substances and under conditions which cause
a risk of explosion, implosion or release of gases. Only operate the Monolith NT.115 with aqueous
solutions.
CAUTION Ensure that the power plug of the external power supply is easily accessible. The Monolith
NT.115 instrument has to be installed in a way that it does not hinder the access to the external
power supply and its power plug.
CAUTION The weight of the Monolith NT.115 instrument is approx. 24 kg, do not move the
instrument alone (two persons required for transport). If you move the instrument alone it poses a
risk of personal injury or damage to the instrument.
CAUTION The manual opening of the instrument is not allowed. Manual opening poses a risk of
personal injury or damage to the instrument. Contact NanoTemper Technologies service personnel
if you need to open the instrument.
CAUTION Use of controls or adjustments or performance of procedures other than those specified
herein may result in hazardous invisible radiation exposure. The instrument contains an IR-laser
module (invisible laser radiation Class 3B according to IEC 60825-1: 2007). Lasers or laser systems
emit intense,coherent electromagnetic radiationthat has the potential of causing irreparable damage
to human skin and eyes. The main hazard of laser radiation is direct or indirect exposure of the eye
to thermal radiation from the infrared B spectral regions (1,400 nm –3,000 nm). Direct eye contact
can cause corneal burns, retinal burns, or both, and possible blindness. Do not open the instrument
while it is switched on. Manual opening of the instrument during operation poses a risk of personal
injury or damage to the instrument. When the instrument is used as intended it is LASER CLASS 1.
CAUTION Mechanical moving parts within the instrument can pinch or injure your hands or fingers.
Do not touch the instrument while parts are moving.
CAUTION The instrument contains a temperature regulator to control the sample temperatures.
Some accessible parts of the instrument can reach temperatures up to 50°C. Avoid touching the
temperature controlled parts of the instrument for a longer time when you have set the temperature
controller to high temperatures.
CAUTION Only NanoTemper Technologies staff is allowed to service and open the instrument. Turn
off the power switch and unplug the power cord before servicing the instrument, unless otherwise
noted. Connect the equipment onlyto the delivered power source. Do not use extension cords. Have
an electrician immediately replace any damaged cords, plugs, or cables. Not doing so poses a risk
of personal injury or damage to the instrument.

5
CAUTION Only open the sample loading site when moving parts inside the instrument are at rest.
Do not insert or remove a sample while linear actuator, laser or LED is at work. Moving parts in the
instrument can be harmful.
CAUTION Do not use the instrument in a cold room.
CAUTION Turn off the main circuit breaker in the back of the chassis, when the instrument is not in
use.
CAUTION Do not use ethanol or other types of organic solvents to clean the instrument as they may
remove the instrument paint.
CAUTION Use the instrument only for biomolecule analytics with aqueous solutions and do not open
the instrument on other site than for sample loading.
CAUTION Only use aqueous sample for analysis in the instrument.
CAUTION Do not use the instrument with hazardous substances or substances/materials which
pose a risk of infections.

6
Regulatory Statement
The following safety and electromagnetic standards were considered:
•IEC 61010-1:2001 Safety requirements for electrical equipment for measurement, control and
laboratory use. Part 1 General Requirements
•IEC 61010-2-010:2005 Safety requirements for electrical equipment for measurement, control
and laboratory use. Part 2-010: Particular requirements for laboratory equipment for the heating
of materials.
•IEC 61010-2-081:2001 Safety requirements for electrical equipment for measurement, control
and laboratory use. Part 2-081: Particular requirements for automatic and semi-automatic
laboratory equipment for analysis and other purposes.
•IEC 60825 Laser safety.
•Complies with 21 CFR 1040.10 and 1040.11 except for deviations pursuant to Laser Notice No.
50, dated June 24, 2007
•IEC 61326-1:2006 EMC, Electrical equipment for measurement, control and laboratory use –
EMC requirements.
•IEC 61000-3-2:2006 EMC, Limits for harmonic current emissions (equipment input current up to
and including 16A per phase).
•IEC 61000-3-3:2008 EMC, Limits

7
Specifications
Input external power supply: 90–264 VAC ± 10% 47–63 Hz, 230 VA max
Output external power supply: 12 VDC, 5.0 A max, 2.4 A typical
Electrical input Monolith NT.115 instrument: 12 VDC, 5A
Environmental
Operating temperature 15 –30°C (Indoor use only)
Storage temperature -20 –30°C
Humidity 0–80%, noncondensing
Operating altitude max 2000m
Monolith NT.115 size
Width 33 cm (13”)
Height 45 cm (17.5”)
Depth 51 cm (20”)
Weight 24 kg (51 lbs) net
Power supply size
Width 10.6 cm (4.2”)
Height 3 cm (1.2”)
Depth 6.5 cm (2.6”)
Power supply weight 0.23 kg (0.51 lbs) net max
IR laser
Wavelength: 1475 nm +/- 15 nm
Power: 120 mW max.
Monolith NT.115 laser classification
Device is LASER PRODUCT CLASS 1
Temperature control
Range: 22°C –45°C (at Room Temperature 25°C)
Accuracy: +/- 0.3°C
Noise level of Monolith NT.115 instrument: max. 64 dB(A)

8
Connections
All ingoing and outgoing connections can be found at the rear panel of the instrument.
Name
Function
Ethernet
Connector for connecting the Ethernet cable to the PC/notebook.
USB
Connector for connecting the USB cable to the PC/notebook. Only
used for service by NanoTemper Technologies service.
Power Switch
When the switch is turned on (position “I”), the instrument is
switched on.
DC Input
The connector to the external power supply. Connect with the
external power supply.

9
Preface
This manual is your guide for using the Monolith NT.115 and doing MicroScale Thermophoresis
(MST) measurements. It instructs first-time users on how to use the instrument, and serves as a
reference for experienced users.
Before using the Monolith NT.115 instrument, please read this instruction manual carefully, and
make sure that the contents are fully understood. This manual should be easily accessible to the
operator at all times during instrument operation. When not using the instrument, keep this manual
in a safe place. If this manual is lost, order a replacement from NanoTemper Technologies GmbH,
by contacting your local NanoTemper Technologies representative.
Notices
1. NanoTemper Technologies shall not be held liable, either directly or indirectly, for any
consequential damage incurred as a result of product use.
2. Prohibitions on the use of NanoTemper Technologies software
•Copying software for other than backup
•Transfer or licensing of the right to use software to a third party
•Disclosure of confidential information regarding software
•Modification of software
•Use of software on multiple workstations, network terminals, or by other methods
3. The contents of this manual are subject to change without notice for product improvement.
4. This manual is considered complete and accurate at publication.
5. This manual does not guarantee the validity of any patent rights or other rights.
6. If a NanoTemper Technologies software program has failed causing an error or improper
operation, this may be caused by a conflict from another program operating on the notebook
(PC). In this case, take corrective action by uninstalling the conflict product(s).
7. NanoTemper Technologies is a registered trademark of NanoTemper Technologies GmbH in
Germany and other countries.

10
Limited Warranty
Products sold by NanoTemper Technologies, unless otherwise specified, are warrantied for a period
of one year from the date of shipment to be free of defects in materials and workmanship. If any
defects in the product are found during this warranty period, NanoTemper Technologies will repair
or replace the defective part(s) or product free of charge.
THIS WARRANTY DOES NOT APPLY TO DEFECTS RESULTING FROM THE FOLLOWING:
1. IMPROPER OR INADEQUATE INSTALLATION.
2. IMPROPER OR INADEQUATE TRANSPORT.
3. IMPROPER OR INADEQUATE OPERATION, MAINTENANCE, ADJUSTMENT OR CALIBRATION.
4. UNAUTHORIZED MODIFICATION OR MISUSE.
5. USE OF UNAUTHORIZED CAPILLARIES AND CAPILLARY TRAYS.
6. USE OF CONSUMABLES, DISPOSABLES AND PARTS NOT SUPPLIED BY AN AUTHORIZED
NANOTEMPER TECHNOLOGIES DISTRIBUTOR.
7. CORROSION DUE TO THE USE OF IMPROPER SOLVENTS, SAMPLES, OR DUE TO
SURROUNDING GASES.
8. ACCIDENTS BEYOND NANOTEMPER TECHNOLOGIES’S CONTROL, INCLUDING NATURAL
DISASTERS.
This warranty does not cover consumables like capillaries, reagents, labeling kits, and the like.
The warranty for all parts supplied and repairs provided under this warranty expires on the warranty
expiration date of the original product. For inquiries concerning repair service, contact NanoTemper
Technologies after confirming the model name and serial number of your NanoTemper Technologies
instrument.

11
Installation Requirements
To ensure operation safety, observe the following conditions:
1. Only operate the Monolith NT.115 instrument with the supplied external power supply (SPU63-
105, Sinpro Electronic Co Ltd or Ael60US12, XP Power LLC or GS90A12-P1M, MeanWell or
GS60A12-P1J, MeanWell).
2. Only connect the external power supply of Monolith NT.115 to an electrical socket containing a
protective conductor terminal.
3. Ensure that the power plug of the external power supply is easily accessible. The Monolith
NT.115 instrument has to be installed in a way that it does not hinder the access to the external
power supply and its power plug.
4. Only operate the instrument with the delivered PC (notebook).
5. Only operate the instrument with original Monolith NT.115 capillary trays.
6. The maximum noise level of the instrument is 64 dB(A). Only operate the instrument in an
environment where this noise level is appropriate.
7. Operate the instrument in a temperature range of 15 –30°C.
8. Operate the instrument in a humidity range of 0 –80 % (RH). If ambient humidity exceeds 80 %
(RH), condensation may deteriorate optical components.
9. Operate the instrument in an atmospheric pressure range of 800 –1060 hPa.
10. Do not operate the Monolith NT.115 under conditions which pose a risk of explosion, implosion
or the release of gases.
11. Avoid strong magnetic fields and sources of high frequency. The instrument may not function
properly when near a strong magnetic field or high frequency source.
12. Avoid vibrations from vacuum pumps, centrifuges, electric motors, processing equipment and
machine tools.
13. Avoid dust and corrosive gas. Do not install the instrument where it may by exposed to dust,
especially in locations exposed to outside air or ventilation outlets.
14. To clean the instrument, only use water.
15. Do not install the instrument in a location where it may be exposed to direct sunlight.
16. Install the instrument in a horizontal and stable position. (This includes a table, bench or desk
upon which the instrument is installed).
17. Ensure that no air conditioner blows air directly onto the instrument. This may prevent stable
measurements.
18. Install the instrument in a location that allows easy access for maintenance.
Note: The above conditions do not guarantee optimal performance of this instrument.

12
Installation and Connecting Cables
Preparation
Prepare a table which can bear a weight of about 50 kg and has a free area of 80 cm (width) x 50 cm
(depth).
Put the Monolith NT.115 instrument and the PC/notebook on this free area.
CAUTION The weight of the Monolith NT.115 instrument is approx. 24 kg, do not move the
instrument alone (two persons required for transport/movement). If you move the instrument alone
it poses a risk of personal injury or damage to the instrument.
Connecting the power supply and the Monolith NT.115
Confirm that the power switch of the Monolith NT.115 instrument is off (power switch is at the back,
left of the instrument).
WARNING! Only operate the Monolith NT.115 instrument with the external power supply provided
(SPU63-105, Sinpro Electronic Co Ltd or Ael60US12, XP Power LLC or GS90A12-P1M, MeanWell
or GS60A12-P1J, MeanWell). Only use the supplied cables and plugs. If not doing so, you risk
electric shock and fire.
Connect the external power supply to the electrical socket. Then connect the power supply to the
Monolith NT.115 instrument.
CAUTION Ensure that the power plug of the external power supply is well accessible. The Monolith
NT.115 instrument has to be installed in a way that it does not hinder the access to the external
power supply and its power plug.
Connect the Monolith NT.115 instrument to the notebook by using the supplied network cable.
Switch on the Monolith NT.115 and the notebook.

13
Maintenance and Operation
Pay attention to the instrument operating environment and always keep it clean so that the
instrument can be used in a stable condition over a long period. Do not place anything on top of the
instrument.
Cleaning the Monolith NT.115 instrument
Switch off the instrument and remove the power plug of the external power supply from the electrical
socket. Only use a dry cloth or a cloth dampened with water for cleaning the instrument.
CAUTION Do not use ethanol or other types of organic solvents to clean the instrument as they may
remove the instrument paint.
Transporting the Monolith NT.115 instrument
Switch off the instrument and remove the power plug of the external power supply from the electrical
socket. Do not carry the instrument alone, two people are needed for transportation.
CAUTION The weight of the Monolith NT.115 instrument is approx. 24 kg, do not move the
instrument alone, two persons required for transport/movement. If you move the instrument alone it
poses a risk of personal injury or damage to the instrument.
Malfunction
In case of a malfunction switch off the Monolith NT.115 instrument and wait for five minutes, then
switch on the instrument again. If the malfunction persists switch off the device, unplug the power
cable and contact the NanoTemper Technologies customer support.
Repairing the Monolith NT.115 instrument
Do not try to repair the instrument. Contact the NanoTemper Technologies customer support for
instrument repairs.
CAUTION The manual opening of the instrument is not permitted. Manual opening poses a risk of
personal injury or damage to the instrument. Contact NanoTemper Technologies customer support
staff if you need to open the instrument.
Waste Treatment
Waste treatment is your own responsibility. You must hand the instrument to a company specialized
in waste recovery. Do not dispose the instrument in a litter bin or at a public waste disposable site.
For detailed information please contact the NanoTemper Technologies service.

14
1. Introduction to MicroScale Thermophoresis
(MST)
MicroScale ThermophoresisTM (MST) provides an easy, fast and precise approach to quantify
biomolecular interactions. It allows for the detection of a wide range of interaction types, ranging
from ion and fragment binding up to interactions of macromolecular complexes such as liposomes
or ribosomes [1-3]. The main key feature of MST is the capability to work under close-to-native
conditions: Measurements are performed immobilization-free and in any buffer, or even in complex
bioliquids such as serum and cell lysate. In addition, the method combines straight forward sample
preparation, low sample consumption of less than 4 μl per sample at nM concentration, and a large
dynamic range with dissociation constants from the sub-nM to mM range. The NanoTemper
Technologies Monolith®instruments are maintenance-free, have low running costs and use an
intuitive software user interface with provides guided assay development and automated data
analysis, making MST a one-of-a-kind, low-hurdle biophysical technology for every lab.
1.1. Method
The Monolith instruments analyze MST signals for rapid and easy determination of affinity constants.
MST is based on the generation of a locally restricted and highly precise temperature change in a
sample by infrared (IR) laser light, which cannot be achieved by conventional heating procedures
using e.g. thermal elements. Changes in sample fluorescence upon activation of the IR laser are
monitored to characterize and quantify binding events. The response of the sample fluorescence
upon IR laser activation is based on distinct physical principles which are described in the following
section.
1.1.1. Underlying physical principles
MicroScale Thermophoresis (MST) is a biophysical technique that measures the strength of the
interaction between two molecules by detecting a variation in the fluorescence signal of a
fluorescently labeled or intrinsically fluorescent target as a result of an IR-laser induced
temperature change. The range of the variation in the fluorescence signal correlates with the
binding of a ligand to the fluorescent target.
Two major factors contribute to the variation in the fluorescence signal:
1- TRIC (Temperature Related Intensity Change) - An effect where the fluorescence intensity of a
fluorophore is temperature dependent. The extent of the temperature dependence is strongly related
to the chemical environment of the fluorophore, which can be changed by the binding of a ligand to
the target.
2- Thermophoresis -An effect where the movement of fluorescent molecules along temperature
gradients results in a quantifiable change in their local concentration and therefore of the observed
fluorescence. The extent of the concentration change depends on the molecule’s overall properties
like size, charge and conformation.
Both TRIC and thermophoresis are influenced by binding events and therefore contribute to the
overall recorded MST signal.

15
In some cases, the fluorescence of the target molecule changes upon ligand binding even in the
absence of an IR-induced temperature gradient. These cases can be analyzed using the
Fluorescence Analysis Mode. The phenomenon is referred to as ligand-induced fluorescence
quenching or enhancement and is typically caused by changes in target conformation or binding of
the ligand in proximity to the target’s fluorophore(s). Since the fluorescence detection in all Monolith®
instruments is highly precise, the change in target fluorescence upon ligand binding can be directly
used to calculate binding affinities without application of the IR laser.
1.1.2. MST Data Analysis
MST experiments can be carried out at different MST intensities, which are defined by the power of
the IR-laser and thus the extent of the temperature change. The higher the MST intensity, the faster
and larger is the temperature increase. During an MST experiment, the sample fluorescence is
recorded, starting with a 3 sec period at ambient temperature to monitor steady-state fluorescence,
followed by IR-laser activation for a defined MST-on time. For data analysis, the change in sample
fluorescence upon IR-laser activation is monitored by calculating the ratio between the fluorescence
after a given MST-on time (F1) and the fluorescence prior to IR laser activation (F0).
1.1.3. Calculation of normalized fluorescence and binding affinities
The observed change in fluorescence after IR laser activation stems from a change in fluorescence
due to TRIC and a change in the concentration of fluorophores due to MST. The overall change in
fluorescence after IR laser activation can thus be expressed as

 󰇛󰇜        
Where 
 describes changes in fluorescence due to TRIC. The thermophoretic change in
concentration is described by 
    The Soret coefficient, ST, describes the percentage of
the concentration change per Kelvin [5].
Measured fluorescence values are displayed as normalized fluorescence (Fnorm) which relates to the
fluorescence values prior (F0)to and after (F1) IR laser activation:
Fnorm=F1/F0
  󰇛󰇜
󰇟󰇠   󰇟󰇠
The latter equation describes the contribution of the bound and unbound state of afluorescent target
molecule (A) to the Fnorm signal. F(A)norm is the contribution of the unbound fluorescent molecule A,
F(AL)norm is the contribution of the complex of the fluorescent molecule A and its interacting ligand L,
and x is the fraction of fluorescent molecules that formed the complex. Upon binding of a ligand to
the target molecule, changes in the MST signal are quantified based on the altered MST signal of
the target-ligand complex. By analyzing a series of samples with an increasing ligand concentration
at constant target concentration, this change in MST signal, expressed as ΔFnorm, can be used to
calculate changes in the fraction of bound target molecule to derive binding affinities.
The dissociation constant, Kd, is obtained by fitting a dose-response curve to a plot of Fnorm vs ligand
concentration. The Kdis calculated from the law of mass action, which is defined as
Kd=[A]*[L]/[AL]

16
where [A] is the concentration of free fluorescent molecule, [L] the concentration of free ligand and
[AL] is the concentration of the complex of A and L. The free concentrations of A and L are [A]=[A0]-
[AL] and [L]=[L0]-[AL], respectively. [A0] is the known concentration of the fluorescent molecule and
[L0] is the known concentration of added ligand. This leads to a quadratic fitting function for [AL]:
[AL]=1/2*(([A0]+[L0]+ Kd)-(([A0]+[L0]+ Kd)2-4*[A0]*[L0])1/2)
The concentration of fluorescent molecule [A0] is kept constant during the experiments and the
concentration of ligand [L0] is varied in a dilution series. The signal obtained in the measurement
directly corresponds to the fraction of fluorescent molecules that formed the complex x=[AL]/[A0],
which can be easily fitted with the derived equation to obtain Kd.
Figure 1: MST setup and experiments. (A) Schematic representation of MST optics. MST is measured in capillaries with
a total volume of down to 4 µl. The fluorescence within the capillary is excited and detected through the same objective. A
focused IR laser is used to locally heat a defined sample volume. The MST response of fluorescent molecules within the
temperature gradient are detected. (B) Typical signal of an MST trace. Initially, the molecules are homogeneously
distributed and a constant ‘‘initial fluorescence’’ is detected. After activation of the IR laser, a drop in fluorescence is
observed, which corresponds to TRIC triggered by the fast temperature change and thermophoretic movement of the
fluorescent molecules out of the heated sample volume. After deactivation of the IR laser, a fluorescence increase occurs,
caused by the ‘backdiffusion’ of molecules and a decrease in sample temperature. MST, MicroScale Thermophoresis; IR,
infrared. (C) Typical binding experiment. The MST signal of a fluorescent molecule (black trace; ‘‘unbound’’) changes upon
binding to a non-fluorescent ligand (grey trace, intermediate state; red trace, ‘‘bound’’), resulting in different traces. (D) For
analysis, the change in MST signal is expressed as the change in the normalized fluorescence (∆Fnorm), which is defined
as F1/F0. Titration of the non-fluorescent ligand results in a gradual change in MST, which is plotted as ∆Fnorm against the
ligand concentration to yield a dose-response curve, which can be fitted to derive binding constants.

17
1.1.4. Software
MST experiments are analyzed using the dedicated MO.Control and MO.Affinity Analysis
Software, using the above equations. Prior to determining binding affinities, the MO.Control software
performs several automated quality checks of the samples to detect protein adsorption,
aggregation, or other irregularities. Based on the results, a guided assay development algorithm
provides detailed instructions on how to efficiently improve assay quality (if necessary). Moreover,
the analysis algorithms automatically determine optimal analysis settings, with the aim of minimizing
exposure times of biomolecules to the IR laser and to limit the temperature increase to < 10 °C. The
optimal binding signal is derived based on the identification of the best signal-to-noise ratio at the
shortest possible MST-on times.
1.2. Literature
1. Jerabek-Willemsen, M., et al., MicroScale Thermophoresis: Interaction analysis and beyond.
Journal of Molecular Structure, 2014(0).
2. Seidel, S.A.I., et al., Label-Free Microscale Thermophoresis Discriminates Sites and Affinity
of Protein–Ligand Binding. Angewandte Chemie International Edition, 2012. 51(42): p.
10656-10659.
3. Wienken, C.J., et al., Protein-binding assays in biological liquids using microscale
thermophoresis. Nat Commun, 2010. 1: p. 100.
4. Duhr, S. and D. Braun, Why molecules move along a temperature gradient. Proc Natl Acad
Sci U S A, 2006. 103(52): p. 19678-82.
5. Baaske, P., et al., Optical thermophoresis for quantifying the buffer dependence of aptamer
binding. Angew Chem Int Ed Engl, 2010. 49(12): p. 2238-41.

18
2. Monolith® NT.115 Instrument
2.1. Models and Most Common Fluorophores
Fluorophore
Excitation
[nm]
NT.115
Blue/Green
NT.115
Blue/Red
NT.115
Green/Red
NT.115
Pico
Emission
[nm]
BCECF
480
525
GFP
488
507
NT-495 (BLUE)
493
521
Fluorescein (FITC)
495
519
Alexa488
495
519
YFP
514
527
Alexa532
530
555
TAMRA
546
579
Cy3
550
570
RFP
555
584
NT-547 (GREEN)
557
574
Alexa546
560
572
Cy5
649
670
NT-647 (RED)
650
670
Alexa647
652
668
Note: Excitation and emission wavelengths can vary depending on the environment (buffer, pH).
Colors are only a very rough approximation. The minimum concentration for use in NT.115 series
instruments can vary.
2.2. Application Range
The Monolith NT.115 allows measurement of the binding of virtually all biomolecules over a large
range of binding affinities (Kd= 1 nM - 500 mM).
2.3. Sensitivity
The Monolith NT.115 can measure as little as the binding of single ions (40 Da) or small molecules
(300 Da) to a target as well as the binding of large complexes such as ribosomes (2.5 MDa).
2.4. Sample Consumption
The Monolith NT.115 requires minimal sample material: typically 1 -10 nM of the labeled interaction
partner and a dilution series of the unlabeled ligand starting about 20-fold above the expected
dissociation constant. For standard applications, 4 μl of sample material is loaded in the capillary.

19
2.5. Capillary Format
The capillary format used for the Monolith NT.115 is inexpensive, easy to handle and offers
maximum flexibility in the experiment scale. The standard sample tray format allows automatic
processing of up to 16 capillaries (e.g. for a detailed Kdanalysis).
2.6. Close-to-Native Conditions
The Monolith NT.115 can monitor the binding affinity between all kinds of biomolecules under close-
to-native conditions:
- immobilization-free
- in a solution of choice, ranging from standard buffers to complex bioliquids including blood
serum, cell lysates, etc.
2.7. Dedicated MO.Control Software and MO.Affinity Analysis Software
The Monolith NT.115 is supported by software for instrument control and data analysis. For more
detailed information please refer to the corresponding software manuals

20
3. Preparation of MST Experiments
3.1. Control Kits - Performing Standard Experiments
Control Kits containing standardized sample material are recommended
- when using the Monolith NT.115 instrument for the first time
- for monitoring the correct performance of the Monolith NT.115
- for training of new laboratory members
Optimized Control Kits for use in MicroScale Thermophoresis are available either with red- or green-
fluorescently labeled biomolecules.
The kits contain a fluorescently labeled DNA molecule, unlabeled binding partner and buffer.
3.2. Selecting the Right Monolith Capillary
NanoTemper Technologies offers coated and uncoated special capillaries with high-precision inner
diameter, best glass quality and verified functionality. Our capillaries are especially developed for
usage with NanoTemper Technologies Monolith devices. It is highly recommended to use
NanoTemper Technologies capillaries for high-quality and reproducible measurements.
NanoTemper Technologies Monolith warranty is only valid if instrument is used with NanoTemper
Technologies capillaries.
Cat #
Monolith Control Kits
Reactions
MO-C030
MonolithTM NT Control Kit RED Molecular Interaction Control Kit
for MonolithTM NT.115 BLUE/RED or MonolithTM NT.115
GREEN/RED
5
MO-C031
MonolithTM NT Control Kit GREEN Molecular Interaction Control
Kit for MonolithTM NT.115 BLUE/GREEN or MonolithTM NT.115
GREEN/RED
5
Cat #
Monolith Capillaries
Content
MO-D001
MonolithTM NT.115 Capillary
Selection Kit
25 Monolith NT.115 Standard Treated Capillaries
25 Monolith NT.115 Hydrophobic Capillaries
25 Monolith NT.115 MST Premium Coated
Capillaries
MO-K002
MonolithTM NT.115 Standard
Treated Capillaries
1000 Capillaries
MO-K003
MonolithTM NT.115
Hydrophobic Capillaries
200 Capillaries
MO-K005
MonolithTM NT.115 MST
Premium Coated Capillaries
200 Capillaries