Thermo Fisher Scientific TrueMark Prostate Protein Assay 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.

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

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

Pelt384_1

Pelt96_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

Thermo Fisher Scientific TrueMark Prostate Protein Assay User guide

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