Premier Products for Superior Life Science Research

NWLSSTM
Thioredoxin Reductase 1
ELISA
Product NWK-TRR01
For Research Use Only

5131 NE 94th Avenue, Suite 201
Vancouver, WA 98662
Phone 360-449-3091 or Toll Free: 888-449-3091
Fax 360-449-3092
E-mail: sales@nwlifescience.com
nwktrr01rev063009

Simple ELISA kit for quantification of humanThioredoxin Reductase 1 (TrxR1) in biologicalsamples.

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To Order Call: 1-888-449-3091

Page

Introduction

3

Intended Use3

Test Principle

3Specifications

4Kit Contents

4

Required Materials Not Provided

4

Required Instrumentation

5Warnings, Precautions, Limitations

5

Storage Instructions

5

Assay Preparation

5

Reagent Preparation

6

Sample Handling/Preparation

7

Standard Curve Preparation

7

Assay Protocol

8

Data Analysis

9

Performance Details

10

References

10

Statement of Limited Warranty

10

Notes

11

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Notes:

Table of Contents

SectionTo Order Call: 1-888-449-3091

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Performance Details:
Specificity
The following substances were tested and found to have no crossreactivity: human TrxR2, mouse TrxR1 and rat TrxR1.

Sensitivity
The minimal detectable dose of human Trx1 was calculated to be
0.39ng/ml, by subtracting two standard deviations from the mean of 10
zero standard replicates (ELISA buffer, S0) and intersecting this value
with the standard curve obtained in the same calculation.
Precision
Intra-assay = 5.7 %Inter-assay = 6.0 %Accuracy:
Recovery on addition from 97.3-101.0%,
Recovery on dilution from 96.0-101.3,

Average = 98.6%
Average = 99.4%

Overall mean recovery = 99.2%
References
1. Mustacich, D. and Powis,G. (2000) Biochem J. 15.346 Pt 1:1-8.
2. Nguyen, P. et al., (2006) Cancer Lett. 18.164-74.
3. Urig, S. and Becker, K. (2006) Semin Cancer Biol. 16. 452-65.
4. Soderberg, A. et al., (2000) Cancer Research 60, 2281-2289
Statement of Limited Warranty:
Northwest Life Science Specialties, LLC (NWLSS) makes no guarantee ofany kind, expressed or implied, that extends beyond the description of thematerial in this kit, except that they will meet our specifications at thetime of delivery. Customer's remedy and NWLSS’ sole liability is limited to,at NWLSS’ option, refund of the purchase price, or the replacement ofmaterial not meeting our specification. By acceptance of our product,
customer assumes all liability and will indemnify and hold NWLSS
harmless for the consequence of this product’s use or misuse by the customer, its employees, or others. Refund or replacement is conditioned ofcustomer notifying NWLSS within twenty-one (21) days of the receipt ofproduct. Failure to give notice within 21 days shall constitute a waiver by
the customer of all claims hereunder with respect to said product.

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Page 3Introduction:
The mammalian thioredoxin reductases (TrxRs) are a family ofselenocysteine containing pyridine nucleotide-disulfide oxido-reductases.
Three different TrxR isoenzymes, TrxR1 as cytosolic, TrxR2 as
mitochondrial, and TrxR3 as testis-specific thiol regulator are known. All
the mammalian TrxRs are homologous to glutathione reductase withrespect to primary structure including the conserved redox catalytic site (Cys-Val-Asn-Val-Gly-Cys-) but distinctively with a C-terminal extension
containing a catalytically active penultimate selenocysteine(SeCys) residue
in the conserved sequence(-Gly-Cys-SeCys-Gly). TrxR is homodimeric protein
in which each monomer includes an FAD prosthetic group, a NADPH bindingsite and a redox catalytic site. Electrons are transferred from NADPH via
FAD and the active-site disulfide to C-terminal SeCys-containing redox
center, which then reduces the substrate like thioredoxin. The members ofTrxR family are 55–58 kDa in molecular size and composed of threeisoforms including cytosolic TrxR1, mitochondrial TrxR2, and TrxR3, also
known as Trx and GSSG reductase (TGR). TrxR plays a key role in protectionof cells against oxidative stress and redox-regulatory mechanism oftranscription factors and various biological phenomena. Many tumor cells
have elevated TrxR levels and TrxR has been shown to play a major role indrug resistance. Inhibition of TrxR and its related redox reactions may thus
contribute to a successful single, combinatory or adjuvant cancer therapy. A
great number of effective natural and synthetic TrxR inhibitors are now
available possessing antitumor potential ranging from induction of oxidative
stress to cell cycle arrest and apoptosis.
Intended Use:
The NWLSS™ Thioredoxin Reductase-1 (TrxR1) ELISA kit is to be used forthe in vitro quantitative determination of human TrxR1 in human serum,
human plasma, cell lysate and buffered solution. The assay will recognize
both native and recombinant human TrxR1.
Test Principle:
The NWLSS™ Thioredoxin Reductase (TrxR1) assay is based on a sandwich
Enzyme-Linked Immunosorbent Assay (ELISA). The microtiter plate
provided in this kit has been pre-coated with a monoclonal antibody specificto human TrxR1. This stationary phase antibody binds sample or standard
TrxR1 while nonbound proteins are removed by washing. Next, boundTrxR1 is tagged with a biotin-conjugated monoclonal antibody specific for
TrxR1 followed by Avidin conjugated to Horseradish Peroxidase (HRP). Subsequent addition of TMB-substrate solution causes blue color (650 nm)
development proportional to the amount of TrxR1 originally captured by thestationary phase antibody. Finally, addition of a sulfuric acid solution stops
the reaction resulting in a yellow color product measured at 450nm. Sample TrxR1 concentration is determined by comparing the 450 nm absorbance of sample wells to the absorbance of known standards.
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Specifications:
Format::To Order Call: 1-888-449-3091

1 X 96 well ELISA presented as 6 X 16 well (2 X 8 well)
Strips in frame.

Number of tests: Triplicate =Duplicate =2440

Specificity:

Human Thioredoxin Reductase 1

Sensitivity:

0.4 ng/mL

Range:

0.4 ng/mL—25ng/mL

Kit Contents:
1 Foil Pouch

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Page 9Assay Protocol: (continued):
16. After appropriate incubation time, add 100ul of Stop Solution to eachwell. The solution in the wells should change from blue to yellow.
17. Read and record the absorbance of each well at 450nm within 20
minutes of adding the Stop Solution.
Data Analysis:
1. Plot the mean absorbance at 450 nm for each standard versus theThioredoxin Reductase 1 concentration. Select the best possible fit forthe curve obtained (4–parameter is recommended). This can typically bedone using the software provided with most plate readers. An example
curve is shown below.

96 well microplate precoated with anti-hu Thioredoxin
Reductase 1

2. Sample Thioredoxin Reductase 1 is determined by comparing theirabsorbance measurements at 450 with those of the standard curve.

1 vialrHu-Thioredoxin Reducatse 1 Standard (lyophilized)

1 bottle

Sample Dilution Buffer(25mL)

3. Sample data as read from the standard curve must be multiplied by thedilution factor used.

1 vial100X Secondary Antibody(Lyophilized Biotin labeled anti-hu TrxR1)(1 Vial)1 bottle

Reagent Dilution Buffer(25mL)1 vial

100X Avidin-HRP Conjugate(150 uL)

1 bottle

Assay Preparation Buffer(30 mL)

1.6

1 bottle

TMB Substrate(20 mL)

1.4

1 bottle

Stop Solution (1 N Sulfuric Acid)(20 mL)

1 bottle

10X Concentrated Wash Buffer(100 mL)

3

Adhesive Plate Covers(3)

Serological pipettes.
Deionized water.

Thioredoxin Reductase
Exam ple Standard Curve

Absorbance at 450 nm

Required Materials Not Provided:
Adjustable micropipettes with disposable tips (5-1000 L). Multi-channelpipettes are useful and help to reduce intra-sample variability.

Note: Samples with an ABS450 exceeding that of the highest standard
should be additionally diluted with Sample Dilution Buffer and re-assayedin order to avoid erroneous results.

1.2
10.8
0.6
R2 = 10.4
0.2
00102030Thioredoxin Reductase Concentration (ng/m L)

Automatic plate washer or other aspiration devices are optional.
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Assay Protocol:
1. Add 300ul of Assay Prep Buffer to all wells and incubate the plate for 5minutes at room temperature.

Required Instrumentation:
Plate reader with 450 nm capability (650 nm is required for optional
monitoring of color development prior to stopping the reaction).

2. Thoroughly aspirate or decant the solution from the wells.

Warnings, Precautions & Limitations:
Reagents are intended for research use only and are not for use indiagnostic or therapeutic procedures.

3. Wash wells 2 times as follows: Dispense 300 uL Working Wash
Solution to each well and allow to soak for 1-3 minutes before decanting
or aspirating the remaining solution from the wells.
4. Add 100ul of Diluted Standards to the appropriate microtiter wells and100ul of Sample Dilution Buffer to zero wells.
5. Add 100ul of Sample to each well according to plan.
6. Cover the plate with the plate cover and incubate for 2 hours at room
temperature.
7. Aspirate or decant the solution from the wells then wash the wells 3
times as previously described in step 3.

Individual components may be harmful if swallowed, inhaled or absorbed
through the skin. Contact should be minimized through the use of gloves
and standard good laboratory practices. If contact with skin or eyes
occurs, rinse the site immediately with water and consult a physician.
Substrate solutions must be at room temperature prior to use. Avoid
contact of substrate solutions with oxidizing agents and metal.
Improper or insufficient washing at any stage of the procedure will
result in either false positive or false negative results. Completely
empty wells before dispensing fresh Wash Buffer, fill with Wash
Buffer as indicated for each wash cycle and do not allow wells to sit
uncovered or dry for extended periods.

8. Add 100ul of Working Secondary Antibody to each well.
9. Cover the plate with the plate cover and incubate for 1 hour at room
temperature (20-25 °C).
10. Aspirate or decant the solution from the wells then wash the wells 3times as previously described in step 3.
11. Add 100ul Working Conjugate Solution to each well.
12. Cover the plate with the plate cover and incubate for 30 minutes atroom temperature (20-25 °C).
13. Thoroughly aspirate or decant the solution from the wells. Wash thewells 3 times previously described in step 3.
14. Add 100ul of TMB Substrate to each well. The liquid in the wells
should begin to turn blue.

15. Incubate the plate at room temperature for approximately 10-15
minutes.
Note: The incubation time for the TMB substrate is dependent on ambientconditions as well as the specific microtiter plate reader in use. The user
should adjust this time as necessary by monitoring the development ofblue color at 650 nm and stopping when the high standard has reachedmaximal absorbance level.
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Storage Instructions:All kit components of this kit are stable at 2 to 8 ℃. Any unusedreconstituted standard should be discarded or frozen at -70 ℃. Standard
can be frozen and thawed one time only without loss of immunoreactivity.
Assay Preparation:
1. Determine the number of wells required to assay standards, samples
and controls for the appropriate number of replicates. It is recommended
that testing be performed in duplicate or triplicate if possible.
2. Create an assay template showing positioning of standards, controls
and samples.
3. Bring all samples and reagents to room temperature before use.
4. To avoid condensation, do not open foil pouches containing the microtiter strips until after they have reached room temperature. Next remove
the required number of strips and place in the frame supplied.
Return unused wells to the storage bag with desiccant, seal and store at
2-8 ºC.

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Reagent Preparation:
Assay Preparation Buffer
The Assay Preparation Buffer is provided ready to use.
Secondary Antibody
1. Reconstitute 100X Secondary Antibody by adding 150 L Reagent
Dilution Buffer to the vial.
2. Equilibrate 100X Secondary Antibody to room temperature, mix gently.
3. Mix 20 l of 100X Secondary Antibody with 2ml Reagent Dilution
Buffer for each 16 well strip to be assayed. Label as “Working SecondaryAntibody Solution”.
4. Return the unused 100X Secondary Antibody to the refrigerator.
AVIDIN-HRP Conjugate
1. Equilibrate to room temperature, mix gently.
2. Mix 20 l of 100X AVIDIN-HRP Conjugate with 2ml Reagent Dilution
Buffer for each16-well strip to be assayed. Label as “Working ConjugateSolution”.
3. Return the unused 100X AVIDIN-HRP Conjugate to the refrigerator.
Wash Buffer
1. Equilibrate to room temperature, mix to re-dissolve any precipitatedsalt.
2. Mix 1 volume 10X Wash Buffer with 9 volumes of deionized water.
Label as “Working Wash Solution”.

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Sample Handling/Preparation
The rate of degradation of native human TrxR1 in various matrices has
not been properly investigated. Therefore, it is beyond the scope of thispublication to comment on specific sample processing protocols or necessary sample dilutional schemes however here are some basic guidelines:
Plasma:Average normal plasma thioredoxin reductase 1 levels have been reported in the range of 18 ng/mL. Therefore, a starting dilution of 2X to 5X
is recommended for plasma from healthy (normal donors). Samples fromoxidative stress or known inflammatory model systems would be expected
may contain significantly elevated levels of TrxR1 such that higher dilutions may be necessary in some situations.
Tissue:Thioredoxin levels are expected to vary greatly in various tissue types
such that proper dilutional schemes for tissue homogenates must beexperimentally determined by the end user.
Standard Curve Preparation:
Reconstitute the human TrxR1 standard to 1g/ml by adding 1ml ofSample Dilution Buffer into the glass vial containing lyophilized human
TrxR1 protein. Swirl or mix gently, and allow to sit for 5 minutes to ensurecomplete reconstitution.
1. Label tubes 1-8 tubes as:25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39 and zero (0) ng/mL.
2. Add 975 L Standard Dilution Buffer to tube 1 and 500 L StandardDilution Buffer to each tube 2-8.
3. Add 25 L Reconstituted 1 ug/mL Standard to tube 1 and mix well.

3. Store both the remaining concentrated Wash Buffer and the Working
Wash Solution at 4°C in the refrigerator.
TMB Substrate
The TMB Substrate is provided ready to use.

4. Perform a serial dilution by transferring 500 L of 25 ng/mL Standard
into tube 2 mixing thoroughly then 500 L of resulting 12.5 ng/mL totube 3 and so on through tube 7 to create all standards down to 0.39 ng/mL.

Stop Solution
The Stop Solution is provided ready to use

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