Nano Temper Prometheus Series User manual

User Manual
Prometheus Series Instruments

2
Content
Safety Considerations ........................................................................................................... 3
Regulatory Statement............................................................................................................ 5
Technical Specifications........................................................................................................ 6
Connections....................................................................................................................... 7
Preface.............................................................................................................................. 8
Notices............................................................................................................................... 8
Limited Warranty................................................................................................................ 9
1. Prometheus Series Instruments ....................................................................................10
1.1. Excitation and Detection for Fluorescence Measurements.....................................10
1.2. Application Range..................................................................................................11
1.3. Sensitivity...............................................................................................................11
1.4. Temperature Range...............................................................................................11
1.5. Precision................................................................................................................11
1.6. Sample Consumption.............................................................................................11
1.7. Capillary Format.....................................................................................................12
1.8. Dedicated Control and Analysis Software...............................................................12
2. nanoDSF Technology....................................................................................................13
3. Using the Prometheus Series Instruments.....................................................................14
3.1. General Usage.......................................................................................................14
3.2. Sample Loading.....................................................................................................14
3.2.1. Sample Loading for the Prometheus NT.48........................................................15
3.2.2. Manual Sample Loading for the Prometheus NT.Plex.........................................15
3.2.3. Automated Sample Loading for the Prometheus NT.Plex...................................16

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Safety Considerations
To ensure operation safety, this instrument must be operated correctly. Carefully read the
following explanations 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, may 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.
CAUTION 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
appropriate steps.
NOTE
ANOTE provides additional information to help the operator
achieve optimal instrument and assay performance.
Read user manual label. This label indicates that you have to
read the user manual before using the instrument. This label is
positioned at the back of the device.
Warning symbol. This symbol indicates a surface that can heat
up and cause burn injuries. This warning label is positioned on
the sample tray.
Warning symbol. This symbol indicates a possible risk for hand
injuries by crushing and sharp edges. This warning label is
positioned on the sample tray.
Identification label. This label is
positioned at the rear panel of the
device.

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WARNING! Operate the Prometheus Series instrument only with the provided external power
supply. Onlyuse the provided cables and plugs. Failure to comply mayresult in a risk of electric
shock and fire.
CAUTION The Prometheus Series instrument has to be installed in a way that does not hinder
access to the external power supply and its power plug.
CAUTION Connect the Prometheus Series instrument power supply in a way that avoids
tripping hazards.
CAUTION Do not use extension cords. Damaged cords, plugs or cables need to be replaced
immediately. Failure to comply may result in a risk of personal injury or damage to the
instrument.
WARNING! Do not operate the Prometheus Series instrument with substances or under
conditions that pose a risk of explosion, implosion or release of gases. Do not use the
instrument with hazardous or infectious substances.
CAUTION Use only aqueous samples for analysis in the instrument.
CAUTION The Prometheus Series instrument weighs approx. 30 kg. Two people are required
for transport. Moving the instrument alone entails a risk of personal injury or damage to the
instrument.
CAUTION Do not open the instrument manually or anywhere other than the sample loading
drawer. Opening entails a risk of personal injury or damage to the instrument and may only be
done by NanoTemper Technologies staff.
CAUTION Only NanoTemper Technologies staff may service the instrument. Turn off the
power switch and unplug the power cord before servicing the instrument, unless instructed
otherwise.
CAUTION The instrument contains a UV-LED. The UV-LED emits invisible ultraviolet radiation
(UVB radiation) when in operation, which may be harmful to eyes and skin, even at brief
periods of exposure. Donot look directly into theUV-LED during operation. If used as intended,
the instrument emits no UV radiation.
CAUTION Mechanical moving parts within the instrument can pinch or injure your hands or
fingers. Do not touch or open the instrument while parts are moving.
CAUTION The display pane is made of glass. Broken glass can injure your hands or fingers.
CAUTION The sharp edges of the Prometheus NT.Plex chipholder thermal element pose a
risk of injury to hands and fingers.
CAUTION The instrument contains a temperature regulator to control the sample temperature.
Some accessible parts of the instrument can reach temperatures of up to 60 °C. Don’t touch
the temperature controlled parts of the instrument when the temperature controller is set to
high temperatures.
CAUTION Do not use the instrument at ambient temperatures below 15 °C.

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CAUTION Use the instrument only at noncondensing conditions (0–80 % humidity, 15-30 °C).
At very high humidity levels, even normal operating temperatures may result in condensation
and corrosion.
CAUTION Turn off the instrument when 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.
Regulatory Statement
The following safety and electromagnetic standards were considered:
•IEC 61010-1:2010 Safety requirements for electrical equipment for measurement,
control and laboratory use. Part 1 General Requirements
•IEC 61010-2-010:2014 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 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

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Technical Specifications
Input of external power supply: 90–264 VAC ± 10 % 47–63 Hz, 230 VA max
Output of external power supply: 24 VDC, 10 A max
Electrical input to Prometheus Series instrument: 24 VDC, 10 A
Environmental:
Operating temperature 15 –30 °C (indoor use only)
Humidity 0–80 %, noncondensing
Operating altitude max 2,000 m
Prometheus Series instrument dimensions:
Width 35 cm (13.8”)
Height 51 cm (20.8”)
Depth 52 cm (20.5”)
Weight 30 kg (66 lbs) net
Power supply dimensions:
Width 21 cm (8.3”)
Height 9 cm (3.5”)
Depth 3 cm (1.1”)
Weight 0.5 kg (1.1 lbs) net max
Temperature control:
Range: 15 °C –95 °C (at 25 °C)
Optional High Temperature Upgrade: 15 °C –110 °C (at 25 °C)
Accuracy: +/- 0.1 °C
Noise level of Prometheus Series instrument: max. 64 dB(A)

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Connections
All ingoing and outgoing connections can be found on the rear panel of the instrument.
Name
Function
Ethernet
Socket for connecting to the PC/laptop via an Ethernet cable.
On/Off Button
Turning the switch to position “I” switches on the instrument.
DC Input
Connector to the external power supply.

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Preface
This manual is a guide for using Prometheus Series instruments and performing nanoDSF
measurements. It instructs first-time users on how to use the instrument, and serves as a
reference for experienced users.
Before using the Prometheus Series instrument, please read this instruction manual carefully,
and make sure that the contents arefully 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.
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 laptop
(PC). In this case, take corrective action by uninstalling the conflicting product(s).
7. NanoTemper is a registered trademark of NanoTemper Technologies GmbH in Germany
and other countries.

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Limited Warranty
Products sold by NanoTemper Technologies, unless otherwise specified, are warrantied to be
free of defects inmaterials and workmanship for a period of one yearfrom the date of shipment.
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 OPERATION, MAINTENANCE, ADJUSTMENT OR
CALIBRATION.
3. UNAUTHORIZED MODIFICATION OR MISUSE.
4. USE OF UNAUTHORIZED CAPILLARIES AND CAPILLARY TRAYS.
5. USE OF CONSUMABLES, DISPOSABLES AND PARTS NOT SUPPLIED BY AN
AUTHORIZED NANOTEMPER DISTRIBUTOR.
6. CORROSION DUE TO THE USE OF IMPROPER SOLVENTS, SAMPLES, OR DUE TO
SURROUNDING GASES.
7. ACCIDENTS BEYOND NANOTEMPER’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
warrantyexpiration date of theoriginal product. For inquiries concerning repair service, contact
NanoTemper Technologies after confirming the model name and serial number of your
NanoTemper Technologies instrument.

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1. Prometheus Series Instruments
The Prometheus Series offers nanoDSF technology (see section 2). nanoDSF is the method
of choice for easy, rapid and accurate analysis of protein stability and aggregation, with
applications in protein engineering, membrane protein research, formulation development and
quality control.
The Prometheus Series comprises two instruments. The Prometheus NT.48 is filled with up to
48 single capillaries for most flexible assay design and easy handling. The Prometheus
NT.Plex employs 24-Capillary chips, which enables manual or automated sample loading. It
can be coupled with robotic and liquid handling platforms for unattended operation and
complete automation.
1.1. Excitation and Detection for Fluorescence Measurements
The Prometheus Series instruments use advanced UV-LEDs for the excitation of fluorophores,
which are superior to UV-lasers in terms of stability, robustness and flexibility. Tryptophan
fluorescence is collected by a proprietary Dual-UV detection unit which is optimized for the
rapid and sensitive acquisition of tryptophan fluorescence at 330 nm and 350 nm.
LED
Fluorophore
Detection
UV (maximum at 285 nm)
Tryptophan, Tyrosine
Dual-UV detector:
330 nm and 350 nm
Detection range:
0-20,000 fluorescence units
The detection unit scansthe sample tray every 3 seconds, recording the fluorescenceintensity
at 330 nm and 350 nm (Figure 1). During a thermal unfolding experiment, the samples are
measured continuously,yielding ultra-high resolution with more than20 data points per minute.
In case the instrument is equipped with the optional Aggregation Optics, scattering information
is collected simultaneously and with equal data point density.
Figure 1: Prometheus high precision capillary format. Capillaries containing 10 µl sample are loaded onto a
thermal element, which can be set to temperatures from 15 °C-95 °C. Within 3 seconds, the whole capillary tray is
read by the Dual-UV detection unit.

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1.2. Application Range
The Prometheus instruments use nanoDSF technology to detect changes in the fluorescence
of the amino acids tryptophan and tyrosine over a wide range of temperatures (see section
1.4). The instruments are used to induce thermal unfolding of proteins and to determine
thermal unfolding transition temperatures. Furthermore, thermal protein refolding can be
monitored and refolding transition temperatures can be determined. The instruments can also
be used for chemical unfolding and refolding experiments.
Optionally, the Prometheus instruments can be equipped with backreflection optics to
investigate aggregation in a sample upon heating and to determine aggregation onset
temperatures.
1.3. Sensitivity
The Prometheus instruments can measure a broad range of concentrations due to an
adjustable LED excitation intensity.
Maximal protein concentration (standard IgG): > 250 mg/ml.
Minimal protein concentration (standard IgG): 5 µg/ml.
1.4. Temperature Range
The thermal element inside Prometheus instruments can be precisely adjusted from 15 °C to
95 °C. Additionally, the instruments can be equipped with a High Temperature Upgrade
allowing a maximum temperature of 110 °C.
Note: If experiments exceed a temperature of 95 °C, or measurement times of 3 hours,
capillaries must be sealed using NanoTemper Technologies Capillary Sealing Paste (PR-
P001) and Capillary Sealing Applicators (PR-P002).
Note: For instruments equipped with a High Temperature Upgrade: When running
experiments with a maximum temperature of 110 °C, it is recommended to use heating rates
of 1 °C/min or slower to ensure linearity of the temperature ramp. For all heating rates, ramp
linearity is guaranteed up to at least 95 °C.
1.5. Precision
Temperature variance across capillary tray in thermal ramp of 1 °C/min: ± 0.2 °C (at 52 °C)
Run-by-run reproducibility of experiments in thermal ramp of 1 °C/min: ± 0.2 °C (at 52 °C)
1.6. Sample Consumption
The Prometheus instruments are designed to minimize the amount of sample needed. Only
10 µl of sample per capillary are required.

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1.7. Capillary Format
The capillary format of the Prometheus instruments is cost-effective, easy to handle and offers
maximal flexibility in the experiment scale, while offering a detection precision that is superior
to other approaches. The number of samples measured in one run can be any number from
one to 48 capillaries in a Prometheus NT.48 instrument, or one to 24 capillaries in a
Prometheus NT.Plex instrument.
1.8. Dedicated Control and Analysis Software
The Prometheus instruments are supported by several software packages to enable straight-
forward measurement setup and data analysis: the PR.ThermControl for thermal unfolding
assays, the PR.ChemControl for chemical unfolding experiments, and the PR.TimeControl for
(iso-)thermal stability analysis and experiments with non-linear temperature ramps or cycles.
Please refer to the individual software manuals for detailed information on the respective kind
of experiment.

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2. nanoDSF Technology
nanoDSF is an advanced Differential Scanning Fluorimetry technology. It detects smallest
changes inthe fluorescence of tryptophan andtyrosine residuespresent invirtually all proteins.
The fluorescence of tryptophans andtyrosines in a protein is strongly dependent ontheir close
surroundings. When located in the hydrophobic core of proteins, tryptophan is shielded from
the surrounding aqueous solvent. Upon unfolding however, tryptophan is exposed, which
alters its photo-physical properties. By following changes in fluorescence, the unfolding of
proteins can be monitored in real time in a truly label-free fashion (Figure 2).
Thermal and chemical unfolding experiments are highly appreciated methods to quantify
protein stability. While thermal unfolding experiments use a temperature ramp to monitor
protein conformational changes, chemical unfolding experiments use chaotropes such as urea
to unfold proteins. Both types of experiments are easily done with the Prometheus Series
instruments.
Figu
The unique dual-UV technology of the Prometheus Series monitors intrinsic tryptophan and
tyrosine fluorescence at the emission wavelengths of 330 nm and 350 nm. To generate an
unfolding curve, either the fluorescence change in one of the two channels, or, alternatively,
the ratio of the fluorescence intensities (F350 nm/F330 nm), is plotted against temperature or
concentration of denaturant (see Figure 2). The fluorescence ratio monitors changes in
fluorescence intensity as well as the tryptophan-specific shift of the fluorescence emission
maximum. It is therefore extremely robust and allows the investigation of virtually all buffer
conditions and even fluorescent additives.
Figure 2: Schematic representation of protein unfolding.
Charged, solvent exposed regions of the protein are colored in
red and blue, the hydrophobic core in gray. Tryptophan residues
are represented as green sticks. The illustration shows their
increasing exposure to the solution upon unfolding. Below, the
respective curve shows the transition from folded to unfolded
protein as a function of temperature or the concentration of a
chemical denaturant.

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3. Using the Prometheus Series Instruments
3.1. General Usage
Start the Prometheus instrument using the power switch at the back left of the instrument, turn
on the control computer and start the software (PR.ThermControl, PR.TimeControl or
PR.ChemControl). It will take approximately one minute for the Prometheus instrument to
complete hardware initialization. You can use the touch display of the Prometheus instrument
to set the internal temperature to a desired value, and to open the capillary tray drawer. These
commands can also be performed in the control software.
After the experimental session is finished, turn the Prometheus instrument off using the switch.
Shut down the control computer. No particular sequence needs to be followed.
Note: No equilibration times are required after startup. Prior to temperature ramps, the
Prometheus instruments automatically perform a one minute equilibration routine.
Note: Always remove capillaries and capillary lids prior to transport of the instrument.
3.2. Sample Loading
Clean the mirrored surface of the capillary tray with a scratch- and dust-free tissue and 99.8 %
ethanol prior to experiments. Keep the tray surface free from dust, dirt and scratches. In order
to detect protein unfolding, proteins must contain at least one tryptophan or other fluorescent
residues that show changes in their fluorescent properties in the dedicated detection range.
10 µl of sample are required per capillary for unfolding experiments. Capillaries (or capillary
chips) are dipped into the sample and automatically fill by capillary forces. Capillary filling is
accelerated by holding the capillary horizontally. Make sure that capillaries are filled
completely. Tolerable capillary filling levels for thermal unfolding experiments are shown in
Figure 3 (top right panel). Once capillaries are filled, place them onto the capillary tray and fix
the capillaries by placing the lid onto the tray. Differences in sample loading between different
Prometheus Series instruments are described in the following sections.
Note: NanoTemper Technologies offers two different types of capillaries, nanoDSF Grade
Standard Capillaries and nanoDSF Grade High Sensitivity Capillaries. To improve sensitivity
at sample concentrations < 200 µg/ml, the nanoDSF Grade High Sensitivity capillaries are
recommended.
Note: Avoid liquid on the outside of the capillary, since this will alter the fluorescence signal. If
there is liquid on the outside of the capillaries, use a dust- and scratch-free tissue to wipe the
capillaries clean.
Note: In case of broken capillaries on the capillary tray, use a paintbrush or tissue to carefully
remove pieces of glass and to avoid scratching the tray.

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3.2.1. Sample Loading for the Prometheus NT.48
The Prometheus NT.48 capillary tray provides 48 numbered slots for single capillaries, offering
maximum flexibility in assay design. It is important that the capillaries are positioned in the
center of the tray. It is also important that the meniscus of the sample does not approach the
heated capillary tray surface, since this will lead to rapid evaporation (Figure 3).
Figure 3: Capillary tray layout and loading for the Prometheus NT.48 instrument. The capillary tray can hold
up to 48 capillaries, which are numbered from 1 to 48, starting in the front closest to the user. When filling the
capillaries, it is important that the capillaries are filled completely, and that they are centered on the capillary tray.
Once all capillaries are loaded onto the capillary tray, place the magnetic lid to fix the capillaries.
3.2.2. Manual Sample Loading for the Prometheus NT.Plex
The Prometheus NT.Plex instrument is designed for fast, automatable high-throughput
measurements. Sample loading can be performed automatically (see 3.2.3) or manually, as
described in this section. The capillaries on Prometheus NT.Plex nanoDSF Grade 24-Capillary
Chips are spaced to enable easy and fast sample loading from 384-well microtiter plates. The
Capillary Chip Filling Station (Cat# NT-AT100) is recommended for manual loading of capillary
chips from microtiter plates since it places the sample at the optimal angle for loading (see

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Figure 4 B). Correct positioning of the capillaries on the tray is ensured by the capillary chip
format.
Place the chip onto the thermal element with the capillaries facing upward (see Figure 4 A).
Use the number imprinted on the chip for orientation, for example to load your sample
number 1. Use the symbols next to the thermal element for correct chip placement. It is
recommended to place the capillary chip with the imprinted number toward the user, since
the capillary closest to the user will be designated “capillary 1” by the software.
The arrow imprinted on the chip indicates the capillary type. An arrow pointing upward (toward
the ) designates standard capillaries while an arrow pointing left (away from the capillaries)
designates high sensitivity capillaries. See 3.2 for recommendations on which capillaries to
use.
Next, place the lid slowly and evenly inside of the guiding structures (see Figure 4 A). The
weight of the lid holds the capillaries in place. Slight movements of the lid (and the capillary
chip) inside of the guiding structures are normal and will not affect the measurement.
CAUTION: Careless, hasty or diagonal placement of the lid can lead to damage of capillaries
or the thermal element. Keep hands away from the instrument when closing the sample
drawer. Especially, be careful not to get caught between capillary lid and instrument, since this
can lead to injury and instrument damage. If necessary, push the ‘Cancel’ button on the touch
display of the instrument to stop the drawer from closing.
Figure 4: Manual sample loading for the Prometheus NT.Plex. (A) When placed correctly, the plastic chip structure
frames the thermal element and the capillaries rest on top of the thermal element. The metal lid is then placed
slowly and evenly inside of the guiding structures, with the rubber padding securing the capillaries in place. The red
arrow indicates the imprinted number on the capillary chip. (B) The Capillary Chip Filling Station is recommended
for manual loading of capillary chips from microtiter plates.
3.2.3. Automated Sample Loading for the Prometheus NT.Plex
The Prometheus NT.Plex instrument can be coupled with the NT.Robotic Autosampler for
automated sample loading. For details, please see the NT.Robotic Autosampler manual.

Contact
NanoTemper Technologies
GmbH
Floessergasse 4
81369 Munich
Germany
Phone: [phone]
Fax: [phone]
[e-mail]
http://www.nanotemper-technologies.com
NanoTemper®, PrometheusTM and nanoDSFTM are
registered trademarks.
V13_2017-0339-133322