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MBS2023542 96 Tests

Enzyme-linked Immunosorbent Assay Kit

For Nuclear Factor Kappa B (NFkB)
Organism Species: Mus musculus (Mouse)
Instruction manual
Version 13.0

[ INTENDED USE ]
The kit is a sandwich enzyme immunoassay for in vitro quantitative measurement of NFkB in mouse tissue
homogenates, cell lysates, cell culture supernates and other biological fluids.

[ REAGENTS AND MATERIALS PROVIDED ]

Reagents Quantity Reagents Quantity

Pre-coated, ready to use 96-well strip plate 1 Plate sealer for 96 wells 4

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Standard 2 Standard Diluent 1×20mL

Detection Reagent A 1×120μL Assay Diluent A 1×12mL

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Detection Reagent B 1×120μL Assay Diluent B 1×12mL

TMB Substrate 1×9mL

ce Stop Solution 1×6mL

Wash Buffer (30 × concentrate) 1×20mL Instruction manual 1

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[ MATERIALS REQUIRED BUT NOT SUPPLIED ]
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1. Microplate reader with 450 ± 10nm filter.

2. Single or multi-channel pipettes with high precision and disposable tips.
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3. Microcentrifuge Tubes.
4. Deionized or distilled water.
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5. Absorbent paper for blotting the microplate.

6. Container for Wash Solution.
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[ STORAGE OF THE KITS ]

1. For unused kit: The whole kit could be stored at -20oC in shelf life, while up to one month at 4oC.
For experiment convenience, reagents could also be stored seperately, Standard, Detection Reagent A,
Detection Reagent B and 96-well strip plate should be stored at -20oC while the others could be at 4 oC.
2. For used kit: When the kit is used, the remaining reagents need to be stored according to the above storage
condition. Besides, please return the unused wells to the foil pouch containing the desiccant pack, and
zip-seal the foil pouch.
Note:
It is highly recommended to use the remaining reagents within 1 month provided this is prior to the expiration date
of the kit. For the expiration date of the kit, please refer to the label on the kit box. All components are stable up to
the expiration date.

[ SAMPLE COLLECTION AND STORAGE ]

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** FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.**
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Tissue homogenates - The preparation of tissue homogenates will vary depending upon tissue type.
1. Tissues were rinsed in ice-cold PBS to remove excess blood thoroughly and weighed before homogenization.
2. Minced the tissues to small pieces and homogenized them in fresh lysis buffer (catalog: MBS2090451, different
lysis buffer needs to be chosen based on subcellular location of the target protein) (w:v = 1:20-1:50, e.g. 1mL
lysis buffer is added in 20-50mg tissue sample) with a glass homogenizer on ice (Micro Tissue Grinders woks,
too).
3. The resulting suspension was sonicated with an ultrasonic cell disrupter till the solution is clarified.
4. Then, the homogenates were centrifuged for 5 minutes at 10,000×g. Collect the supernates and assay
immediately or aliquot and store at ≤-20oC.
Cell Lysates - Cells need to be lysed before assaying according to the following directions.
1. Adherent cells should be washed by cold PBS gently, and then detached with trypsin, and collected by
centrifugation at 1,000×g for 5 minutes (suspension cells can be collected by centrifugation directly).
2. Wash cells three times in cold PBS.

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3. Resuspend cells in fresh lysis buffer with concentration of 107 cells/mL. If it is necessary, the cells could be
subjected to ultrasonication till the solution is clarified.
4. Centrifuge at 1,500×g for 10 minutes at 2-8oC to remove cellular debris. Assay immediately or aliquot and

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store at ≤-20oC.
Cell culture supernates and other biological fluids - Centrifuge samples for 20 minutes at 1,000×g. Collect the
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supernates and assay immediately or store samples in aliquot at -20oC or -80oC for later use. Avoid repeated
freeze/thaw cycles.
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Note:
1. Samples to be used within 5 days may be stored at 4oC, otherwise samples must be stored at -20oC (≤1
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month) or -80oC (≤2 months) to avoid loss of bioactivity and contamination.

2. Sample hemolysis will influence the result, so hemolytic specimen should not be used.
3. When performing the assay, bring samples to room temperature.
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[ REAGENT PREPARATION ]
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1. Bring all kit components and samples to room temperature (18-25oC) before use. If the kit will not be used up
in one time, please only take out strips and reagents for present experiment, and leave the remaining strips
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and reagents in required condition.

2. Standard - Reconstitute the Standard with 1.0mL of Standard Diluent, kept for 10 minutes at room
temperature, shake gently(not to foam). The concentration of the standard in the stock solution is 20ng/mL.
Please firstly dilute the stock solution to 10ng/mL and the diluted standard serves as the highest standard
(10ng/mL). Then prepare 7 tubes containing 0.5mL Standard Diluent and use the diluted standard to produce
a double dilution series according to the picture shown below. Mix each tube thoroughly before the next
transfer. Set up 7 points of diluted standard such as 10ng/mL, 5ng/mL, 2.5ng/mL, 1.25ng/mL, 0.625ng/mL,
0.312ng/mL, 0.156ng/mL, and the last EP tubes with Standard Diluent is the blank as 0ng/mL.

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** FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.**
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Tube 1 2 3 4 5 6 7 8 9

ng/mL 20 10 5 2.5 1.25 0.625 0.312 0.156 0

3. Detection Reagent A and Detection Reagent B - Briefly spin or centrifuge the stock Detection A and
Detection B before use. Dilute them to the working concentration 100-fold with Assay Diluent A and B,
respectively.
4. Wash Solution - Dilute 20mL of Wash Solution concentrate (30×) with 580mL of deionized or distilled water
to prepare 600mL of Wash Solution (1×).
5. TMB substrate - Aspirate the needed dosage of the solution with sterilized tips and do not dump the residual

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solution into the vial again.
Note:
1. Making serial dilution in the wells directly is not permitted.

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2. Prepare standards within 15 minutes before assay. Please do not dissolve the reagents at 37oC directly.
3. Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction,
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and avoid foaming and mix gently until the crystals are completely dissolved. To minimize imprecision caused
by pipetting, use small volumes and ensure that pipettors are calibrated. It is recommended to suck more
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than 10μL for one pipetting.
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4. The reconstituted Standards, Detection Reagent A and Detection Reagent B can be used only once.
5. If crystals have formed in the Wash Solution concentrate (30×), warm to room temperature and mix gently
until the crystals are completely dissolved.
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6. Contaminated water or container for reagent preparation will influence the detection result.
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[ SAMPLE PREPARATION ]
1. We are only responsible for the kit itself, but not for the samples consumed during the assay. The user should
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calculate the possible amount of the samples used in the whole test. Please reserve sufficient samples in
advance.
2. Please predict the concentration before assaying. If values for these are not within the range of the standard
curve, users must determine the optimal sample dilutions for their particular experiments. Sample should be
diluted by PBS.
3. If the samples are not indicated in the manual, a preliminary experiment to determine the validity of the kit is
necessary.
4. Tissue or cell extraction samples prepared by chemical lysis buffer may cause unexpected ELISA results due
to the impacts from certain chemicals.
5. Due to the possibility of mismatching between antigen from other origin and antibody used in our kits (e.g.,
antibody targets conformational epitope rather than linear epitope), some native or recombinant proteins
from other manufacturers may not be recognized by our products.
6. Influenced by the factors including cell viability, cell number or sampling time, samples from cell culture
supernates may not be detected by the kit.
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** FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.**
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7. Fresh samples without long time storage is recommended for the test. Otherwise, protein degradation and
denaturalization may occur in those samples and finally lead to wrong results.

[ ASSAY PROCEDURE ]
1. Determine wells for diluted standard, blank and sample. Prepare 7 wells for standard, 1 well for blank.
Add 100μL each of dilutions of standard (read Reagent Preparation), blank and samples into the appropriate
wells. Cover with the Plate sealer. Incubate for 1 hour at 37oC.
2. Remove the liquid of each well, don’t wash.
3. Add 100μL of Detection Reagent A working solution to each well, cover the wells with the plate sealer and
incubate for 1 hour at 37oC.
4. Aspirate the solution and wash with 350μL of 1× Wash Solution to each well using a squirt bottle,
multi-channel pipette, manifold dispenser or autowasher, and let it sit for 1~2 minutes. Remove the remaining
liquid from all wells completely by snapping the plate onto absorbent paper. Totally wash 3 times. After the
last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against
absorbent paper.

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5. Add 100μL of Detection Reagent B working solution to each well, cover the wells with the plate sealer and
incubate for 30 minutes at 37oC.
6. Repeat the aspiration/wash process for total 5 times as conducted in step 4.

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7. Add 90μL of Substrate Solution to each well. Cover with a new Plate sealer. Incubate for 10 - 20 minutes at
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37oC (Don't exceed 30 minutes). Protect from light. The liquid will turn blue by the addition of Substrate
Solution.
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8. Add 50μL of Stop Solution to each well. The liquid will turn yellow by the addition of Stop solution. Mix the
liquid by tapping the side of the plate. If color change does not appear uniform, gently tap the plate to ensure
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thorough mixing.
9. Remove any drop of water and fingerprint on the bottom of the plate and confirm there is no bubble on the
surface of the liquid. Then, run the microplate reader and conduct measurement at 450nm immediately.
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Note:
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1. Assay preparation: Keep appropriate numbers of wells for each experiment and remove extra wells from
microplate. Rest wells should be resealed and stored at -20oC.
：Please use the freshly prepared Standard. Please carefully add samples
2. Samples or reagents addition：
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to wells and mix gently to avoid foaming. Do not touch the well wall. For each step in the procedure, total
dispensing time for addition of reagents or samples to the assay plate should not exceed 10 minutes. This
will ensure equal elapsed time for each pipetting step, without interruption. Duplication of all standards and
specimens, although not required, is recommended. To avoid cross-contamination, change pipette tips
between additions of standards, samples, and reagents. Also, use separated reservoirs for each reagent.
3. Incubation: To ensure accurate results, proper adhesion of plate sealers during incubation steps is
necessary. Do not allow wells to sit uncovered for extended periods between incubation steps. Once
reagents are added to the well strips, DO NOT let the strips DRY at any time during the assay. Incubation
time and temperature must be controlled.
4. Washing: The wash procedure is critical. Complete removal of liquid at each step is essential for good
performance. After the last wash, remove any remaining Wash Solution by aspirating or decanting and
remove any drop of water and fingerprint on the bottom of the plate. Insufficient washing will result in poor
precision and false elevated absorbance reading.

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** FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.**
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5. Controlling of reaction time: Observe the change of color after adding TMB Substrate (e.g. observation once
every 10 minutes), if the color is too deep, add Stop Solution in advance to avoid excessively strong reaction
which will result in inaccurate absorbance reading.
6. TMB Substrate is easily contaminated. Please protect it from light.
7. The environment humidity which is less than 60% might have some effects on the final performance, therefore, a
humidifier is recommended to be used at that condition.
[ TEST PRINCIPLE ]
The microplate provided in this kit has been pre-coated with an antibody specific to NFkB. Standards or samples are
then added to the appropriate microplate wells with a biotin-conjugated antibody specific to NFkB. Next, Avidin
conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate
solution is added, only those wells that contain NFkB, biotin-conjugated antibody and enzyme-conjugated Avidin will
exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the
color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of NFkB in the
samples is then determined by comparing the O.D. of the samples to the standard curve.
[ CALCULATION OF RESULTS ]

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Average the duplicate readings for each standard, control, and samples and subtract the average zero standard optical
density. Create a standard curve with NFkB concentration on the y-axis and absorbance on the x-axis. Draw a best fit
curve through the points and it can be determined by regression analysis. If samples have been diluted, the

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concentration read from the standard curve must be multiplied by the dilution factor.
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[ TYPICAL DATA ]
In order to make the calculation easier, we plot the O.D. value of the standard (X-axis) against the known concentration
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of the standard (Y-axis), although concentration is the independent variable and O.D. value is the dependent variable.
However, the O.D. values of the standard curve may vary according to the conditions of assay performance (e.g.
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operator, pipetting technique, washing technique or temperature effects), plotting log of the data to establish standard
curve for each test is recommended. Typical standard curve below is provided for reference only.
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Typical Standard Curve for NFkB, Mouse ELISA.

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** FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.**
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[ DETECTION RANGE ]
0.156-10ng/mL. The standard curve concentrations used for the ELISA’s were 10ng/mL, 5ng/mL, 2.5ng/mL,
1.25ng/mL, 0.625ng/mL, 0.312ng/mL, 0.156ng/mL.

[ SENSITIVITY ]
The minimum detectable dose of NFkB is typically less than 0.057ng/mL.
The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration
that could be differentiated from zero. It was determined by adding two standard deviations to the mean optical
density value of twenty zero standard replicates and calculating the corresponding concentration.

[ SPECIFICITY ]
This assay has high sensitivity and excellent specificity for detection of NFkB.
No significant cross-reactivity or interference between NFkB and analogues was observed.
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Limited by current skills and knowledge, it is impossible for us to complete the cross- reactivity detection between
NFkB and all the analogues, therefore, cross reaction may still exist.

[ PRECISION ]

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Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level NFkB were tested 20
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times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level NFkB were tested
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on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
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Intra-Assay: CV<10%
Inter-Assay: CV<12%
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[ STABILITY ]
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The stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% prior to
the expiration date under appropriate storage condition.
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To minimize extra influence on the performance, operation procedures and lab conditions, especially room
temperature, air humidity, incubator temperature should be strictly monitored. It is also strongly suggested that
the assay is performed by the same operator from the beginning to the end.

[ SAMPLE VALUE ]
Serum/Plasma - Thirty-five serum and plasma samples from specific pathogen free (SPF) mouse were
evaluated in this assay. All samples measured less than the lowest NFkB standard.
Tissue Homogenates - Tissues from specific pathogen free (SPF) mouse were prepared according to the
protocol shown in [ SAMPLE COLLECTION AND STORAGE ] section on page 2. Aliquots of the lysates were
assayed for NFkB levels. Results are shown in the table below.
Samples(Tissue) O.D. Range

Heart 1.15-1.56
Uterus 0.53-0.82
These data are our in-house data, only for reference.
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** FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.**
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[ ASSAY PROCEDURE SUMMARY ]

1. Prepare all reagents, samples and standards;
2. Add 100μL standard or sample to each well. Incubate 1 hour at 37oC;
3. Aspirate and add 100μL prepared Detection Reagent A. Incubate 1 hour at 37oC;
4. Aspirate and wash 3 times;
5. Add 100μL prepared Detection Reagent B. Incubate 30 minutes at 37oC;
6. Aspirate and wash 5 times;
7. Add 90μL Substrate Solution. Incubate 10-20 minutes at 37oC;
8. Add 50μL Stop Solution. Read at 450nm immediately.
[ IMPORTANT NOTE ]
1. Limited by the current conditions and scientific technology, we can't completely conduct the comprehensive
identification and analysis on the raw material provided by suppliers. So there might be some qualitative and
technical risks to use the kit.
2. The final experimental results will be closely related to validity of the products, so the kit should be used prior to the

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expiration date. And please store the kits exactly according to the instruction.
3. Kits from different batches may be a little different in detection range, sensitivity and color developing time. Please
perform the experiment exactly according to the instruction attached in kit while electronic ones from our website is

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only for reference.
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Do not mix or substitute reagents from one kit lot to another. Use only the reagents supplied by manufacturer.
5. Protect all reagents from strong light during storage and incubation. All the bottle caps of reagents should be
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covered tightly to prevent the evaporation and contamination of microorganism. TMB Substrate should remain
colorless till it is reacted with the enzyme which binds to the microplate.
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6. There may be some foggy substance in the wells when the plate is opened at the first time. It will not have any
effect on the final assay results. Do not remove microplate from the storage bag until needed.
7. Wrong operations during the reagents preparation and loading, as well as incorrect parameter setting for the plate
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reader may lead to incorrect results. A microplate reader with a bandwidth of 10nm or less and an optical density
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range of 0-3 O.D. at 450 ± 10nm wavelength is acceptable for use in absorbance measurement. Please read the
instruction carefully and adjust the instrument prior to the experiment.
8. Variation in sample preparation and each step of experimental operation may cause different results. In order to get
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better reproducible results, the operation of each step in the assay should be controlled.
9. Each kit has been strictly passed Q.C test. However, results from end users might be inconsistent with our in-house
data due to some unexpected transportation conditions or different lab equipments. Intra-assay variance among
kits from different batches might arise from above factors, too.
10. Kits from different manufacturers with the same item might produce different results, since we haven’t compared
our products with other manufacturers.
11. The standard of the kit and immunogen used for antibody preparation are commonly recombinant proteins, as
different fragments, expression systems, purification methods might be used in recombinant protein preparation,
we can not guarantee the kit could detect recombinant protein from other companies. So, it is not recommended to
use the kit for the detection of recombinant protein.
12. Please predict the concentration of target molecules in samples, or arrange a preliminary experiment, it is a good
way to solve specific problem, e.g. the concentration of samples are beyond the detection range of the kit.
13. The kit might not be suitable for detection of samples from some special experiment, for instance, knock-out

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** FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.**
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experiments, due to their uncertainty of effectiveness.
14. The instruction manual is also for the kit of 48T, but all reagents of 48T kit are reduced by half.
15. The kit is designed for research use only, we will not be responsible for any issue if the kit was used in clinical
diagnostic or any other procedures.

[ PRECAUTION ]
The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection
when using this material.

[ TROUBLE SHOOTING ]
Problem Possible Source Correction Action
Poor Improper standard curve preparation Ensure accurate operation of the dilution
Standard Incomplete washing and aspiration Adequate washing and adequate aspiration
Curve Inaccurate Pipetting Check and Calibrate pipettes
Incomplete washing of wells Ensure sufficient washing

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Poor Inadequate mixing and aspiration reagents Adequate aspiration and mixing reagents
Precision Reused pipette tips, containers and sealers Change and use new pipette tips, containers and sealers

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Inaccurate Pipetting Check and Calibrate pipettes
Inadequate reagent volumes added to wells Calibrate pipettes and Add adequate reagents
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Incorrect incubation times Ensure sufficient incubation times
Low
Incorrect incubation temperature Reagents balanced to room temperature
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O.D
Conjugate or substrate reagent failure Mix conjugate & substrate, color should develop immediately
Values
No stop solution added Follow the assay protocol in the kit manual
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Read beyond suggested reading time Read within the time recommended in the manual
Improper Sample Storage Store the sample properly and use the fresh sample
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Sample
Improper sample collection and preparation Take proper sample collection and preparation method
Values
Low quantity of analyte in samples Use new sample and repeat assay
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** FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.**