Thermo Fisher Scientific TrueMark Prostate Protein Assay User guide

Brand
Thermo Fisher Scientific
Model
TrueMark Prostate Protein Assay
Type
User guide
For Research Use Only. Not for use in diagnostic procedures.
TrueMark™ Prostate Protein Assay
USER GUIDE
Detection of KLK2, PSP94, FPSA, PSA, and GDF15 in human
plasma research samples
for use with:
Biomek™ FXP Automated Workstation
Biomek™ FX Automated Workstation
QuantStudio™ 12K Flex Real–Time PCR System
Catalog Number A46360
Publication Number MAN0017766
Revision B.0
Life Technologies Corporation | 6055 Sunol Blvd | Pleasanton, CA 94566
For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE
LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR
ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Revision history: Pub. No. MAN0017766
Revision Date Description
B.0 6 March 2020 Updated product names and made minor edits for style or clarication.
A.0 22 April 2019 New document for Early Access users.
Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these
products, you accept the terms and conditions of all applicable Limited Use Label Licenses.
TRADEMARKS: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
©2020 Thermo Fisher Scientific Inc. All rights reserved.
Contents
■Product information ....................................................... 5
Product description ............................................................. 5
Contents and storage ............................................................ 6
Required materials not supplied ................................................... 7
Optimize tip usage .......................................................... 9
Procedure overview ............................................................ 10
How the TrueMark™ Prostate Protein Assay works ............................. 10
Research workow ........................................................ 11
■Methods ................................................................... 15
Procedural guidelines ........................................................... 15
Day 1—Prepare the Sample Stocks Plate ......................................... 15
Day 1—Set up, then run the Plate 1 binding reaction on the Biomek™ FXP instrument ... 16
Before you begin .......................................................... 16
Set up the instrument deck ................................................. 17
Run Binding Reaction Setup Method—Plate 1, then incubate for binding reaction . 21
Day 1—Set up, then run the Plate 2 binding reaction on the Biomek™ FXP instrument ... 22
Before you begin .......................................................... 22
Set up the instrument deck ................................................. 22
Run Binding Reaction Setup Method—Plate 2, then incubate for binding reaction . 26
Day 2—Set up, then run Ligation Reaction Setup Method on the Biomek™
FXP instrument ................................................................ 27
Before you begin .......................................................... 27
Set up the instrument deck ................................................. 27
Run Ligation Reaction Setup Method ........................................ 30
Day 2—Set up, then run the ligation reaction on the ProFlex™ thermal cycler .......... 30
Day 2—Set up, then run the real‑time PCR on the QuantStudio™ 12K Flex Instrument .. 31
Generate the plate setup les ............................................... 31
Run the real‑time PCR ...................................................... 32
Evaluate protein concentration and data quality with TaqMan™ Protein
Concentration Software ......................................................... 33
Overview of TaqMan™ Protein Concentration Software ......................... 33
Open the experiment le in TaqMan™ Protein Concentration Software ............ 33
Review results ............................................................. 34
TrueMark™ Prostate Protein Assay User Guide 3
Approve the results and export to a LIMS (optional) ............................ 35
Export the results table ..................................................... 35
■APPENDIX A Troubleshooting ......................................... 36
Biomek™ FXP instrument ........................................................ 36
TrueMark™ Prostate Protein Assay performance ................................... 38
Good laboratory practices for PCR and RT-PCR ................................... 41
■APPENDIX B Biomek™ FXP instrument automated
pipetting workow ........................................................ 42
Day 1—Plate 1 binding reactions (KLK2, PSP94) ................................... 42
Day 1—Plate 2 binding reactions (FPSA, PSA, GDF15) .............................. 43
Day 2—Plate 1/Plate 2 ligation reaction ........................................... 44
■APPENDIX C Biomek™ FXP instrument orientation .................. 45
Biomek™ FXP instrument deck layout ............................................. 45
Initialize the Biomek™ FXP instrument ............................................. 45
Biomek™ FXP instrument light curtain ............................................. 47
Biomek™ FXP maintenance schedule ............................................. 47
■APPENDIX D Safety .................................................... 49
Chemical safety ................................................................ 50
Biological hazard safety ......................................................... 52
■Documentation and support ............................................ 53
Related documentation ......................................................... 53
Customer and technical support ................................................. 53
Limited product warranty ....................................................... 54
Contents
4TrueMark™ Prostate Protein Assay User Guide
Product information
IMPORTANT! Before using this product, read and understand the information in the
“Safety” appendix in this document.
Product description
The TrueMark™ Prostate Protein Assay kit enables protein expression analysis in
human plasma research samples of ve proteins of interest in prostate cancer
research. The kit includes all reagents, assay probes, and calibrator proteins for
sensitive, specic, and reproducible assessment of protein expression of the
following proteins:
•Human kallikrein 2 (KLK2)
•Prostate secretory protein 94 (PSP94)
•Free prostate-specic antigen (FPSA)
•Total prostate-specic antigen (PSA)
•Growth/differentiation factor 15 (GDF15)
The kit supports a combination of plasma samples and a positive control in a
384-well plate format. The fully automated research workow is compatible with the
Biomek™ FXP and Biomek™ FX instruments.
The TrueMark™ Prostate Protein Assay uses TaqMan™ Protein Assays technology.
The technology combines the sensitive, specic protein-binding capabilities of
antibodies with the quantication capabilities of 5′‑nuclease real-time PCR (see “How
the TrueMark™ Prostate Protein Assay works” on page 10). TaqMan™ Protein Assays
enable identication and quantication of proteins from initial sample volumes as
small as 2.5 µL in a single, homogeneous assay format.
TrueMark™ Prostate Protein Assay User Guide 5
Contents and storage
Table 1 TrueMark™ Prostate Protein Assay (Cat. No. A46360; assays sufficient for
56 individual samples)
Contents Amount Storage
Day 1 Binding—KLK2, PSP94 (Plate 1)
Sample Buffer 7.5 mL
−25°C to −15°C
Do not store in a
frost‑free freezer.
Probe Buffer (white cap) 2 × 1.5 mL
PSP94 Probe A (purple cap) 12 µL
PSP94 Probe B (purple cap) 12 µL
PSP94 Calibrator (purple cap) 12 µL
PSP94 Buffer (tan cap) 800 µL
KLK2 Probe A (blue cap) 2 × 12 µL
KLK2 Probe B (blue cap) 2 × 12 µL
KLK2 Calibrator (blue cap) 2 × 12 µL
Day 1 Binding—FPSA, PSA, GDF15 (Plate 2)
Sample Buffer 7.5 mL
−25°C to −15°C
Do not store in a
frost‑free freezer.
Probe Buffer (white cap) 2 × 1.5 mL
FPSA Probe A (orange cap) 12 µL
FPSA Probe B (orange cap) 12 µL
FPSA Calibrator (orange cap) 12 µL
PSA Probe A (teal cap) 12 µL
PSA Probe B (teal cap) 12 µL
PSA Calibrator (teal cap) 12 µL
GDF15 Probe A (yellow cap) 12 µL
GDF15 Probe B (yellow cap) 12 µL
GDF15 Calibrator (yellow cap) 12 µL
Day 2 Ligation—qPCR
Ligation-qPCR Master Mix 2 × 13.5 mL −25°C to −15°C
Do not store in a
frost‑free freezer.
DNA Ligase (natural cap) 2 × 70 µL
Master Mix Additive (red cap) 2 × 185 µL
Product information
Contents and storage
6TrueMark™ Prostate Protein Assay User Guide
Note: The kit is also available in combination with the TrueMark™ Prostate Positive
Control (Cat. No. A47026).
Required materials not supplied
Unless otherwise indicated, all materials are available through thermofisher.com.
"MLS" indicates that the material is available from fisherscientific.com or another
major laboratory supplier.
Table 2 Instruments
Item Source
Biomek™ FXP Automated Workstation with Dual
Arm System, Multichannel Pipettor and Span-8
Pipettors
Methods for the workow:
•Binding Reaction Setup Method—Plate 1
•Binding Reaction Setup Method—Plate 2
•Ligation Reaction Setup Method
Beckman Coulter A31844
ProFlex™ 2 × 384‑well PCR System 4484077
QuantStudio™ 12K Flex Real–Time PCR System,
384-well block Contact your local sales ofce
Table 3 Software
Item Source
TaqMan™ Protein Concentration Software Contact your local eld application
scientist (FAS)
Table 4 Reagents
Item Source
TrueMark™ Prostate Positive Control A47025[1]
[1] Also available in combination with the TrueMark™ Prostate Protein Assay (Cat. No. A47026)
Product information
Required materials not supplied
TrueMark™ Prostate Protein Assay User Guide 7
Table 5 Equipment and accessories
Item Source
Plate centrifuge, capable of 2,800 × g at 4°C MLS
Frame for Modular Reservoirs Beckman Coulter 372795
24-Position Tube Rack, 1 rack (2 ‑mL rack) Beckman Coulter 373661
Insert, 11 mm, for 1.5-mL Microfuge Tubes
(11‑mm insert),13 inserts Beckman Coulter 373696
MicroAmp™ 96-Well Base, 3 bases (96‑well base) N8010531
Benchtop microcentrifuge MLS
MicroAmp™ Optical Film Compression Pad, 2
pads 4312639
Static Peltier ALP, 3 Peltiers Beckman Coulter A93938
Aluminum Tube Block (0.5‑mL rack) Beckman Coulter A83054
1×1 Static ALP, 6 ALPs Beckman Coulter B87477
1×3 Static ALP, 3 ALPs Beckman Coulter B87478
MicroAmp™ Adhesive Film Applicator 4333183
Table 6 Plates, tips, and consumables (sufficient for 56 plasma samples)
Item Amount Source
MicroAmp™ EnduraPlate™ Optical
384-Well Clear Reaction Plates with
Barcode (384‑well EnduraPlate™)
2 plates 4483285
MicroAmp™ Fast Optical 96-Well
Reaction Plate, 0.1 mL (Fast 96‑well
plate)
8 plates 4346907
Nunc™ 1.0 mL DeepWell™ Plates
(sterile) (1.0‑mL deep-well plate) 2 plates 260251
AP96 P20 Tips (light blue) 11 boxes Beckman Coulter 717255
Span-8 P20 Tips (blue) 8 boxes[1] Beckman Coulter 379506
Span-8 P250 Tips (green) 5 boxes[1] Beckman Coulter 379503
Span-8 P1000 Tips (orange) 4 boxes[1] Beckman Coulter B01124
MicroAmp™ Clear Adhesive Film 4 sealing lms 4306311
Product information
Required materials not supplied
8TrueMark™ Prostate Protein Assay User Guide
Item Amount Source
MicroAmp™ Optical Adhesive Film 2 sealing lms 4360954
Quarter Reservoir, Divided by
Length 4 reservoirs 372788
[1] See “Optimize tip usage” on page 9.
The Biomek™ FXP methods do not consume the entire box of tips during each run.
After each method, you can transfer tips to the empty holes in the boxes, to optimize
tip usage.
Table 7 Optimized tip usage on the Biomek™ FXP instrument
Tip type Maximum usage Optimized usage
Span-8 P20 Tips 8 boxes 5 boxes
Span-8 P250 Tips 5 boxes 2 boxes
Span-8 P1000 Tips 4 boxes 1 box
Optimize tip
usage
Product information
Required materials not supplied
TrueMark™ Prostate Protein Assay User Guide 9
Procedure overview
The TrueMark™ Prostate Protein Assay uses TaqMan™ Protein Assays technology.
TaqMan™ Protein Assays use a matched pair of target-specic antibodies, each
conjugated to a DNA oligonucleotide. This technology leverages the sensitivity and
large dynamic range of TaqMan™ real-time PCR. During antibody-analyte binding, the
two DNA oligonucleotides are brought into close proximity, which allows ligation of
the two strands and subsequent creation of the template strand for amplication.
Binding Ligation Real-time PCR
Assay probe B
Assay probe A
Protein target
Cycle number
ΔRn
Assay probes A and B are
introduced to the test and
reference samples. Each
probe consists of a single-
stranded oligonucleotide with
a streptavidin-biotin linkage
to an antibody that
recognizes an epitope of the
target protein.
When combined in solution,
the antibody portions of the
probes bind to the
corresponding epitopes on
the target protein.
A third oligonucleotide
(shown in green) is
introduced that hybridizes to
the oligonucleotide ends of
the assay probes. The hybrid
structure forms preferentially
when the assay probes are
bound to the same protein
and are consequently in
proximity to each other.
DNA ligase then covalently
links the hybridized ends of
the assay probes to form a
continuous DNA molecule
linking the bound antibodies.
The samples undergo real-
time PCR amplication that
specically targets ligated
assay probes.
High target concentration
Intermediate target
concentration
Low target concentration
Background
How the
TrueMark™
Prostate
Protein Assay
works
Product information
Procedure overview
10 TrueMark™ Prostate Protein Assay User Guide
The TrueMark™ Prostate Protein Assay workow uses the Biomek™ FXP instrument
for automated setup of binding, ligation, and real-time PCR in the same 384-well
plate. The Ligation-qPCR Master Mix, DNA Ligase, and Master Mix Additive enable
ligation and real-time PCR to be performed sequentially in the sealed plate without
further intervention. The ligation reaction is performed on the ProFlex™ System, then
the 384-well plate is transferred to the QuantStudio™ 12K Flex Instrument for
real‑time PCR. TaqMan™ Protein Concentration Software calculates protein
concentrations from the real-time PCR results that are obtained with the
QuantStudio™ 12K Flex Software.
Day 1—prepare the Sample Stocks Plate
EDTA plasma samples
▼
Manually transfer aliquots of plasma test samples ( ) plus the TrueMark™ Prostate
Positive Control ( ) to a Fast 96‑well plate
Note: User-dened control samples can be used in place of test samples.
▼
(Fast 96
‑
well plate)
Sample Stocks Plate
Day 1—binding reaction
Plate 1 binding reactions (KLK2, PSP94)
(Fast 96
‑
well plate) (Reservoir frame with quarter
reservoir)
(0.5
‑
mL rack)
A
B
C
D
1 2 3 4 56
A
B
C
D
1 2 3 4 56
(2
‑
mL rack with inserts)
Sample Stocks Plate Sample Buffer Probes (left) and
Calibrators (right)
(KLK2, PSP94)
2 × Probe Buffer,
PSP94 Buffer
▼
Run Binding Reaction Setup Method—Plate 1 (~50 minutes)
(See Appendix B, “Biomek™ FXP instrument automated pipetting workow”)
Research
workflow
Product information
Procedure overview
TrueMark™ Prostate Protein Assay User Guide 11
▼
(Fast 96
‑
well plate)
Sample Dilutions Plate
▼
Proceed to set up Plate 2 binding reactions
▼
1 2 3 4
5
6
7
8
9 10 11 12 13
14
15 16 17 18
19
20 21 22 23 24
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
(384
‑
well EnduraPlate™)
Plate 1 (samples + Probes + Calibrators)
▼
>16 hours, 4°C (binding reaction)
▼
Proceed to Day 2—Plate 1 ligation and real-time PCR
Plate 2 binding reactions (FPSA, PSA, GDF15)
(start after Binding Reaction Setup Method—Plate 1 is complete)
(Fast 96
‑
well plate) (Reservoir frame with quarter
reservoir)
(0.5
‑
mL rack)
A
B
C
D
1 2 3 4 56
(2
‑
mL rack with inserts)
Sample Dilutions Plate Sample Buffer Probes (left) and
Calibrators (right)
(FPSA, PSA,
GDF15)
2 × Probe Buffer
▼
Run Binding Reaction Setup Method—Plate 2 (~45 minutes)
(See Appendix B, “Biomek™ FXP instrument automated pipetting workow”)
▼
1 2 3 4
5
6
7
8
9 10 11 12 13
14
15 16 17 18
19
20 21 22 23 24
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
(384
‑
well EnduraPlate™)
Plate 2 (samples + Probes + Calibrators)
▼
>16 hours, 4°C (binding reaction)
▼
Proceed to Day 2—Plate 2 ligation and real-time PCR
Product information
Procedure overview
12 TrueMark™ Prostate Protein Assay User Guide
Day 2—ligation and real-time PCR
Plate 1 (KLK2, PSP94)
IMPORTANT! Keep Plate 2 at 4°C during Plate 1 ligation and real‑time PCR.
1 2 3 4
5
6
7
8
9 10 11 12 13
14
15 16 17 18
19
20 21 22 23 24
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
(384
‑
well EnduraPlate™)(Reservoir frame with quarter reservoir) (0.5
‑
mL rack)
Plate 1
(probe binding to Plate 1 samples
complete)
Ligation-qPCR Master Mix DNA Ligase, Master Mix
Additive
▼
Run Ligation Reaction Setup Method (~16 minutes)
(See Appendix B, “Biomek™ FXP instrument automated pipetting workow”)
▼
1 2 3 4
5
6
7
8
9 10 11 12 13
14
15 16 17 18
19
20 21 22 23 24
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Plate 1 (ligation reaction complete)
▼
Set up, then run the ligation reaction on the ProFlex™ thermal cycler (page 30)—ProFlex™ System
▼
Run the real‑time PCR (page 32)—QuantStudio™ 12K Flex Instrument
Plate 2 (FPSA, PSA, GDF15)
1 2 3 4
5
6
7
8
9 10 11 12 13
14
15 16 17 18
19
20 21 22 23 24
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
(384
‑
well EnduraPlate™)(Reservoir frame with quarter reservoir) (0.5
‑
mL rack)
Plate 2
(probe binding to Plate 2 samples
complete)
Ligation-qPCR Master Mix DNA Ligase, Master Mix
Additive
Product information
Procedure overview
TrueMark™ Prostate Protein Assay User Guide 13
▼
Run Ligation Reaction Setup Method (~16 minutes)
(See Appendix B, “Biomek™ FXP instrument automated pipetting workow”)
▼
1 2 3 4
5
6
7
8
9 10 11 12 13
14
15 16 17 18
19
20 21 22 23 24
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Plate 2 (ligation reaction complete)
Set up, then run the ligation reaction on the ProFlex™ thermal cycler (page 30)—ProFlex™ System
▼
Run the real‑time PCR (page 32)—QuantStudio™ 12K Flex Instrument
Product information
Procedure overview
14 TrueMark™ Prostate Protein Assay User Guide
Methods
Procedural guidelines
•Handle plasma samples according to applicable guidelines and regulations.
•Perform all centrifugation steps as described in the procedure.
•Place each 11‑mm insert in the designated locations of the 24-Position Tube
Rack before placing a tube in the insert. Ensure that the insert is ush with the
rack.
•See the Biomek™ Liquid Handler User’s Manual for proper use of the instrument.
•This procedure is optimized for processing on the Biomek™ FXP and Biomek™ FX
instruments.
•The methods run on the Biomek™ FXP instrument require specied deck layout
and pod names. See “Biomek™ FXP instrument deck layout” on page 45.
•Before starting a run on the Biomek™ FXP instrument, place a biohazardous
waste bag in the tip waste location.
•The Biomek™ FXP instrument must be homed before the rst run of the day. See
“Initialize the Biomek™ FXP instrument” on page 45.
•The Peltiers must be initialized and allowed to reach set temperature before the
start of the procedure. See “Initialize the Biomek™ FXP instrument” on page 45.
•Do not reach across the Biomek™ FXP instrument light curtain while the arms are
in motion. See “Biomek™ FXP instrument light curtain” on page 47.
Day 1—Prepare the Sample Stocks Plate
Before starting this procedure, collect EDTA plasma samples according to your
laboratory methodology and keep frozen until use.
IMPORTANT! Do not dilute samples before use.
1. Thaw plasma test samples and the TrueMark™ Prostate Positive Control as
indicated.
•For small volumes ≤100 µL, thaw the samples on ice or at 4°C until
completely thawed.
•For large volumes of plasma test samples in 4‑mL or 8‑mL tubes, thaw the
samples at room temperature with gentle rotation to allow for even mixing.
Note: User‑dened control samples can be used instead of test samples.
TrueMark™ Prostate Protein Assay User Guide 15
2. Briey vortex test and control samples for 10–15 seconds, then centrifuge the
samples at 1,400 × g for 30 seconds to collect the contents at the bottom of the
tubes.
3. Label the side of a Fast 96‑well plate "Sample Stocks", then place the plate in a
96‑well base on ice.
4. Transfer each test or control sample
to the Sample Stocks Plate,
following the layout shown.
•Test samples—transfer 30 µL
(recommended volume; the
volume can range from a
minimum of 15 µL up to 50 µL).
•TrueMark™ Prostate Positive
Control—transfer 20 µL to each of 2 wells of the Sample Stocks Plate.
Note: A minimum of 1 positive control well should be run on each plate.
Note: Columns 8–12 are not used in the methods. Do not place samples in
those wells.
5. Seal the plate with a MicroAmp™ Clear Adhesive Film.
6. Centrifuge the Sample Stocks Plate at 2,800 × g for 10 minutes at 4°C.
Keep the plate at 4°C or on ice until use.
Proceed to the next step, “Day 1—Set up, then run the Plate 1 binding reaction on
the Biomek™ FXP instrument” on page 16, within 30 minutes.
Do not store or reuse the Sample Stocks Plate.
Day 1—Set up, then run the Plate 1 binding reaction on the
Biomek™ FXP instrument
•Ensure that the Biomek™ FXP instrument is homed and the Peltiers are at set
temperature before the rst run of the day (see “Initialize the Biomek™ FXP
instrument” on page 45).
•Approximately 30 minutes before starting this procedure, thaw the following
reagents at room temperature.
–2 tubes of Probe Buffer
–1 tube of PSP94 Buffer
–1 bottle of Sample Buffer
•After the reagents have thawed, keep at 4°C or on ice until use.
•Store Probes and Calibrators at −20°C until use.
Sample Stocks Plate layout.
Test samples
TrueMark™ Prostate Positive Control
Before you
begin
Methods
Day 1—Set up, then run the Plate 1 binding reaction on the Biomek™ FXP instrument
16 TrueMark™ Prostate Protein Assay User Guide
1. Gently mix the Probe Buffer and PSP94 Buffer by inversion 10–15 times, briey
centrifuge the tubes to collect the contents at the bottom of the tubes, then
place on ice until use.
2. Gently mix the Sample Buffer by inversion 10–15 times, then place on ice until
use.
3. In the Biomek™ software, open Binding Reaction Setup Method—Plate 1 to
view the deck layout. For the completed deck setup, also see Figure 1 on
page 20.
4. Set up empty plates on the instrument deck.
Table 8 Plate setup for Binding Reaction Setup Method—Plate 1
Plate Deck position
1.0‑mL deep‑well plate P6
Fast 96‑well plate on 96‑well base P9
Fast 96‑well plate Pelt96_1
Fast 96‑well plate, labeled "Sample Dilutions Plate" on the side,
on 96‑well base
P11
384‑well EnduraPlate™, labeled "Plate 1" on the side Pelt384_1
5. Set up boxes of tips on the deck, then remove their lids.
Table 9 Tips setup for Binding Reaction Setup Method—Plate 1
Tips Deck position
AP96 P20 Tips (light blue) TL1
AP96 P20 Tips (light blue) P1
Span-8 P250 Tips (green) P4
Span-8 P250 Tips (green) P5
Span-8 P1000 Tips (orange) P8
Span-8 P20 Tips (blue) P13
Span-8 P20 Tips (blue) P14
Span-8 P20 Tips (blue) P15
Set up the
instrument
deck
Methods
Day 1—Set up, then run the Plate 1 binding reaction on the Biomek™ FXP instrument
TrueMark™ Prostate Protein Assay User Guide 17
6. Set up the 2 ‑mL rack with Probe Buffer and PSP94 Buffer.
a. Place three 11-mm inserts in
positions A6, B6, and C6 of a 24-
Position Tube Rack.
b. Uncap the two tubes of thawed
Probe Buffer and rmly place the
tubes in positions A6 and B6.
c. Uncap the tube of PSP94 Buffer
and rmly place the tube in
position C6.
d. Place the 2 ‑mL rack in position P10 of the instrument deck, with row D
nearest to the front of the instrument (see Figure 1 on page 20).
7. Set up the quarter reservoir with
Sample Buffer.
a. Add the entire bottle volume of
thawed Sample Buffer to one
side of a Quarter Reservoir,
Divided by Length, then place
the reservoir in a Frame for Modular Reservoirs so that the liquid side of the
reservoir is at the outermost edge of the frame.
b. Place the reservoir-frame assembly in position P7 of the instrument deck,
so that the liquid is on the left and outermost edge of the frame (see
Figure 1 on page 20).
8. Remove the adhesive lm from the Sample Stocks Plate, place it on a 96‑well
base, then place the plate/base assembly in position P12 of the instrument
deck.
9. Set up the 0.5‑mL rack with Probes and Calibrators.
Note: The 0.5‑mL rack is already on the instrument deck (see Figure 1 on
page 20).
a. Remove Probes A and B and Calibrators for PSP94 and KLK2 (two tubes)
from storage, then briey centrifuge to collect the contents at the bottom of
the tubes.
IMPORTANT! Do not vortex Probes or Calibrators.
A
B
C
D
1 2 3 4 56
A
B
C
D
1 2 3 4 56
2 ‑mL rack layout.
2 × Probe Buffer
PSP94 Buffer
Reservoir frame with quarter reservoir.
Methods
Day 1—Set up, then run the Plate 1 binding reaction on the Biomek™ FXP instrument
18 TrueMark™ Prostate Protein Assay User Guide
b. Uncap, then place each tube as indicated in the following table.
Table 10 0.5‑mL rack insert layout for Probes and Calibrators
123456
AKLK2
Probe A
KLK2
Probe B Empty KLK2
Calibrator Empty Empty
BKLK2
Probe A
KLK2
Probe B Empty KLK2
Calibrator Empty Empty
CPSP94
Probe A
PSP94
Probe B Empty PSP94
Calibrator Empty Empty
D Empty Empty Empty Empty Empty Empty
10. Ensure that all reagents, plates, samples, and tips are in the correct location on
the instrument deck (see Figure 1 on page 20).
Methods
Day 1—Set up, then run the Plate 1 binding reaction on the Biomek™ FXP instrument
TrueMark™ Prostate Protein Assay User Guide 19
A
B
C
D
1 2 3 4 56
TL1
P1
P2
P3
P4
P6
P7
1
2
3
4
5
6
7
8
9
10
11
12
A
B
C
D
E
F
G
H
P9
1
2
3
4
5
6
7
8
9
10
11
12
A
B
C
D
E
F
G
H
P12
P8
1
2
3
4
5
6
7
8
9
10
11
12
A
B
C
D
E
F
G
H
P11
P13
P14
P15
WTR1
1 2 3 4
5
6
7
8
9 10 11 12 13
14
15 16 17 18
19
20 21 22 23 24
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Pelt384_1
P5 Pelt96_1
P10 PeltFlat_1
A
B
C
D
1 2 3 4 56
A
B
C
D
1 2 3 4 56
Figure 1 Biomek™ FXP deck setup for Binding Reaction Setup Method—Plate 1
AP96 P20 Tips
AP96 P20 Tips
Span-8 P250 Tips
Span-8 P250 Tips
1.0‑mL deep-well plate
Sample Buffer (quarter reservoir in frame, liquid on the
left)
Span-8 P1000 Tips
Fast 96‑well plate on 96‑well base
2 ‑mL rack (2 × Probe Buffer, PSP94 Buffer)
Empty Sample Dilutions Plate (Fast 96‑well plate) on
96‑well base
TL1
P1
P4
P5
P6
P7
P8
P9
P10
P11
Empty Plate 1 ( 384‑well EnduraPlate™)
Fast 96‑well plate
0.5‑mL rack (Probes A and B, Calibrators for KLK2,
PSP94)
Sample Stocks Plate on 96‑well base
Liquid waste
Tip waste
Span-8 P20 Tips
Span-8 P20 Tips
Span-8 P20 Tips
Pelt384_1
PeltFlat_1
P12
W
TR1
P13
P14
P15
Methods
Day 1—Set up, then run the Plate 1 binding reaction on the Biomek™ FXP instrument
20 TrueMark™ Prostate Protein Assay User Guide
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Thermo Fisher Scientific TrueMark Prostate Protein Assay User guide

Brand
Thermo Fisher Scientific
Model
TrueMark Prostate Protein Assay
Type
User guide
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