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含WW域E3泛素蛋白连接酶2(WWP2)检测试剂盒(酶联免疫吸附试验法)
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联&系&人：华夏远洋
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所&在&地&址：安徽合肥
详&细&地&址：滨湖万科城4#1605
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产品详细说明
USCN(Cloud-Clone Corp)
中国?境内关外
Serum, plasma, tissue homogenates, cell lysates, cell culture supernates and other biological fluids.
Homo sapiens (Human，人)
WB, ICC, IHC-P, IHC-F, ELISA
检测方法：
酶联免疫吸附试验法
0.781-50ng/mL
ELISA Kit for WW Domain Containing E3 Ubiquitin Protein Ligase 2 (WWP2)
SEL087Hu 96 Tests&
Enzyme-linked Immunosorbent Assay Kit&
For WW Domain Containing E3 Ubiquitin Protein Ligase 2 (WWP2)&
Organism Species: Homo sapiens (Human)&
Instruction manual&
FOR IN VITRO AND RESEARCH USE ONLY&
NOT FOR USE IN CLINICAL DIAGNOSTIC PROCEDURES&
11th Edition (Revised in July, 2013)&
[ INTENDED USE ]&
The kit is a sandwich enzyme immunoassay for in vitro quantitative measurement of WWP2 in human tissue&
homogenates, cell lysates 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&
Standard 2 Standard Diluent 1&20mL&
Detection Reagent A 1&120&L Assay Diluent A 1&12mL&
Detection Reagent B 1&120&L Assay Diluent B 1&12mL&
TMB Substrate 1&9mL Stop Solution 1&6mL&
Wash Buffer (30 & concentrate) 1&20mL Instruction manual 1&
[ MATERIALS REQUIRED BUT NOT SUPPLIED ]&
1. Microplate reader with 450 & 10nm filter.&
2. Precision single or multi-channel pipettes and disposable tips.&
3. Eppendorf Tubes for diluting samples.&
4. Deionized or distilled water.&
5. Absorbent paper for blotting the microtiter plate.&
6. Container for Wash Solution&
[ STORAGE OF THE KITS ]&
1. For unopened kit: All the reagents should be kept according to the labels on vials. The Standard, Detection&
Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20oC upon receipt while&
the others should be at 4 oC.&
2. For opened kit: When the kit is opened, the remaining reagents still 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 reseal along entire edge of zip-seal.&
It is highly recommended to use the remaining reagents within 1 month provided this is within 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 until&
this expiration date.&
[ SAMPLE COLLECTION AND STORAGE ]&
Tissue homogenates - The preparation of tissue homogenates will vary depending upon tissue type. For this&
assay, tissues were rinsed in ice-cold PBS(0.01mol/L,pH 7.0-7.2) to remove excess blood thoroughly and&
weighed before homogenization. Minced the tissues to small pieces and homogenized them in 5-10mL of&
PBS with a glass homogenizer on ice(Micro Tissue Grinders woks, too). The resulting suspension was&
sonicated with an ultrasonic cell disrupter or subjected to two freeze-thaw cycles to further break the cell&
membranes. After that, the homogenates were centrifugated for 5 minutes at 5000&g. Remove the supernate&
and assay immediately or aliquot and store at & -20oC.&
Cell Lysates - Cells must be lysed before assaying according to the following directions.&
1. Adherent cells should be detached with trypsin and then collected by centrifugation (suspension cells can&
be collected by centrifugation directly).&
2. Wash cells three times in cold PBS.&
3. Resuspend cells in PBS (1&) and the cells was subject to ultrasonication for 4 times (or Freeze cells at &
-20oC. Thaw cells with gentle mixing. Repeat the freeze/thaw cycle for 3 times.)&
4. Centrifuge at 1500&g for 10 minutes at 2 - 8oC to remove cellular debris.&
Other biological fluids - Centrifuge samples for 20 minutes at 1000&g. Remove particulates and assay&
immediately or store samples in aliquot at -20oC or -80oC. Avoid repeated freeze/thaw cycles.&
1. Samples to be used within 5 days may be stored at 4oC, otherwise samples must be stored at -20oC (&1&
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 detected.&
3. When performing the assay, bring samples to room temperature.&
[ REAGENT PREPARATION ]&
1. Bring all kit components and samples to room temperature (18-25oC) before use.&
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 100ng/mL.&
Please firstly dilute the stock solution to 50ng/mL and the diluted standard serves as the highest standard&
(50ng/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 50ng/mL, 25ng/mL, 12.5ng/mL, 6.25ng/mL, 3.12ng/mL,&
1.56ng/mL, 0.78ng/mL, and the last EP tubes with Standard Diluent is the blank as 0ng/mL. &
3. Detection Reagent A and Detection Reagent B - Briefly spin or centrifuge the stock Detection A and&
Detection B before use. Dilute to the working concentration 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&
solution into the vial again.&
1. Making serial dilution in the wells directly is not permitted.&
2. Prepare standard 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,&
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&
than 10&L for once pipetting.&
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.&
6. Contaminated water or container for reagent preparation will influence the detection result.&
[ SAMPLE PREPARATION ]&
1. Cloud-Clone Corp. is only responsible for the kit itself, but not for the samples consumed during the assay.&
The user should 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.&
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.&
Tube 1 2 3 4 5 6 7 8 9&
ng/mL 100 50 25 12.5 6.25 3.12 1.56 0.78 0 &
6. Influenced by the factors including cell viability, cell number or sampling time, samples from cell culture&
supernatant may not be detected by the kit.&
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 100&L each of dilutions of standard (read Reagent Preparation), blank and samples into the appropriate&
wells. Cover with the Plate sealer. Incubate for 2 hours at 37oC.&
2. Remove the liquid of each well, don&t wash.&
3. Add 100&L of Detection Reagent A working solution to each well. Incubate for 1 hour at 37oC after covering&
it with the Plate sealer.&
4. Aspirate the solution and wash with 350&L 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.&
5. Add 100&L of Detection Reagent B working solution to each well. Incubate for 30 minutes at 37oC after&
covering it with the Plate sealer.&
6. Repeat the aspiration/wash process for total 5 times as conducted in step 4.&
7. Add 90&L of Substrate Solution to each well. Cover with a new Plate sealer. Incubate for 15 - 25 minutes at&
37oC (Don't exceed 30 minutes). Protect from light. The liquid will turn blue by the addition of Substrate&
Solution.&
8. Add 50&L 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&
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.&
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.&
2. Samples or reagents addition：Please use the freshly prepared Standard. Please carefully add samples&
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.&
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 microtiter plate provided in this kit has been pre-coated with an antibody specific to WWP2. Standards or&
samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to&
WWP2. 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 WWP2, 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 WWP2 in the samples is then determined by comparing the&
O.D. of the samples to the standard curve.&
[ CALCULATION OF RESULTS ]&
Average the duplicate readings for each standard, control, and samples and subtract the average zero standard&
optical density. Construct a standard curve by plotting the mean O.D. and concentration for each standard and&
draw a best fit curve through the points on the graph or create a standard curve on log-log graph paper with&
WWP2 concentration on the y-axis and absorbance on the x-axis. Using some plot software, for instance, curve&
expert 1.30, is also recommended. If samples have been diluted, the concentration read from the standard curve&
must be multiplied by the dilution factor.&
[ TYPICAL DATA ]&
In order to make the calculation easier, we plot the O.D. value of the standard (X-axis) against the known&
concentration 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. 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. &
Typical Standard Curve for WWP2, Human ELISA.&
[ DETECTION RANGE ]&
0.78 -50ng/mL. The standard curve concentrations used for the ELISA&s were 50ng/mL, 25ng/mL, 12.5ng/mL,&
6.25ng/mL, 3.12ng/mL, 1.56ng/mL, 0.78ng/mL.&
[ SENSITIVITY ]&
The minimum detectable dose of WWP2 is typically less than 0.27ng/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 WWP2.&
No significant cross-reactivity or interference between WWP2 and analogues was observed.&
Limited by current skills and knowledge, it is impossible for us to complete the cross- reactivity detection between&
WWP2 and all the analogues, therefore, cross reaction may still exist.&
[ PRECISION ]&
Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level WWP2 were tested&
20 times on one plate, respectively.&
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level WWP2 were tested&
on 3 different plates, 8 replicates in each plate.&
CV(%) = SD/meanX100&
Intra-Assay: CV&10%&
Inter-Assay: CV&12%&
[ STABILITY ] &
The stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within&
the expiration date under appropriate storage condition.&
To minimize extra influence on the performance, operation procedures and lab conditions, especially room&
temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the&
whole assay is performed by the same operator from the beginning to the end.&
[ ASSAY PROCEDURE SUMMARY ]&
1. Prepare all reagents, s&
2. Add 100&L standard or sample to each well. Incubate 2 hours at 37oC;&
3. Aspirate and add 100&L prepared Detection Reagent A. Incubate 1 hour at 37oC;&
4. Aspirate and wash 3&
5. Add 100&L prepared Detection Reagent B. Incubate 30 minutes at 37oC;&
6. Aspirate and wash 5&
7. Add 90&L Substrate Solution. Incubate 15-25 minutes at 37oC;&
8. Add 50&L Stop Solution. Read at 450nm immediately.&
[ IMPORTANT NOTE ]&
1. Limited by the current condition 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, operation skills of the end&
users and the experimental environments. Please make sure that sufficient samples are available.&
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 only for information.&
4. 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&
covered tightly to prevent the evaporation and contamination of microorganism.&
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 microtiter plate from the storage bag until needed.&
7. Wrong operations during the reagents preparation and loading, as well as incorrect parameter setting for the&
plate reader may lead to incorrect results. A microplate plate reader with a bandwidth of 10nm or less and an&
optical density range of 0-3 O.D. or greater at 450 & 10nm wavelength is acceptable for use in absorbance&
measurement. Please read the instruction carefully and adjust the instrument prior to the experiment. For&
more information, please refer to the operation video&
8. Even the same operator might get different results in two separate experiments. In order to get better&
reproducible results, the operation of every step in the assay should be controlled. Furthermore, a&
preliminary experiment before assay for each batch is recommended.&
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 instruction manual also suits for the kit of 48T, but all reagents of 48T kit are reduced by half.&
[ 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&
Improper standard curve preparation Ensure accurate operation of the dilution&
Incomplete washing and aspiration Adequate washing and adequate aspiration&
Inaccurate Pipetting Check and Calibrate pipettes&
Precision&
Incomplete washing of wells Ensure sufficient washing&
Inadequate mixing and aspiration reagents Adequate aspiration and mixing reagents&
Reused pipette tips, containers and sealers Change and use new pipette tips, containers and sealers&
Inaccurate Pipetting Check and Calibrate pipettes&
Inadequate reagent volumes added to wells Calibrate pipettes and Add adequate reagents&
Incorrect incubation times Ensure sufficient incubation times&
Incorrect incubation temperature Reagents balanced to room temperature&
Conjugate or substrate reagent failure Mix conjugate &substrate,color should develop immediately&
No stop solution added Follow the assay protocol in the kit manual&
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&
Improper sample collection and preparation Take proper sample collection and preparation method&
Low quantity of analyte in samples Use new sample and repeat assay&
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含WW域E3泛素蛋白连接酶2(WWP2)检测试剂盒(酶联免疫吸...相关产品报价
公司名称报价日期
VIP北京华夏远洋科技有限公司
中国/美国/德国
VIP上海信裕生物科技有限公司
中国?境内关外
VIP北京华夏远洋科技有限公司
VIP上海钰博生物科技有限公司
VIP上海沪震实业有限公司
VIP上海钰博生物科技有限公司
1元/96T/48T
VIP上海康朗生物科技有限公司
中国?境内关外
VIP北京华夏远洋科技有限公司
美国/德国/中国
VIP上海谷研科技有限公司
美国/德国/中国
VIP上海谷研科技有限公司
欧洲、美国、德国、美国
VIP上海邦景实业有限公司
上海抚生实业有限公司
上海抚生实业有限公司
上海抚生实业有限公司
欧美、德国、中国
上海一研生物科技有限公司
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