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1. gt Cloud Clone Corp SEB364Hu 96 Tests Enzyme linked Immunosorbent Assay Kit For Breast Cancer Susceptibility Protein 2 BRCA2 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 BRCA2 in human tissue homogenates cell lysates and other biological fluids REAGENTS AND MATERIALS PROVIDED Pre coated ready to use 96 well strip plate Plate sealer for 96 wells Standard Standard Diluent 1x20mL Detection Reagent B 1x120uL Assay Diluent B 1x12mL TMB Substrate Stop Solution 1x6mL Wash Buffer 30 x concentrate 1x20mL Instruction manual MATERIALS REQUIRED BUT NOT SUPPLIED 1 Microplate reader with 450 10nm filter Detection Reagent A 1x120uL Assay Diluent A 1x12mL 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 20 C upon receipt while the others should be at 4 C 2 For opened kit When the kit is
2. 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 Resuspend cells in PBS 1x and the cells was subject to ultrasonication for 4 times or Freeze cells at lt 20 C Thaw cells with gentle mixing Repeat the freeze thaw cycle for 3 times 4 Centrifuge at 1500xg for 10 minutes at 2 8 C to remove cellular debris Other biological fluids Centrifuge samples for 20 minutes at 1000xg Remove particulates and assay immediately or store samples in aliquot at 20 C or 80 C Avoid repeated freeze thaw cycles Note 1 Samples to be used within 5 days may be stored at 4 C otherwise samples must be stored at 20 C lt 1 month or 80 C lt 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 25 C 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 10ng mL Please prepare 7 tubes containing 0 5mL Standard D
3. TX TTO84 USA 001 BERS 60 7402 weewclood clone os mailia cheud clone us Export Processing fone Wuhan Hubei 430056 PRC 00 6 800 880 4087 www clowd clone com mail cloud clonc com CD Cloud Clone Corp 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 samples and standards 2 Add 100uL standard or sample to each well Incubate 2 hours at 37 C 3 Aspirate and add 100uL prepared Detection Reagent A Incubate 1 hour at 37 C 4 Aspirate and wash 3 times 5 Add 100uL prepared Detection Reagent B Incubate 30 minutes at 37 C 6 Aspirate and wash 5 times 7 Add 90uL Substrate Solution Incubate 15 25 minutes at 37 C 8 Add 50uL Stop Solution Read at 450nm immediately IMPORTANT NOTE a 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 ri
4. 26 Houston TX TTOR4 USA 00 S740 www clood clone os maila choud clone us Export Processing Zone Wuhan Hubei 430046 PRC 0086 800 880 0687 wew cloud clonc com mail cloud clonc com gt Cloud Clone Corp 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 Influenced by the factors including cell viability cell number or sampling time samples from cell culture supernatant may not be detected by the kit 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 100uL 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 37 C Remove the liquid of each well don t wash Add 100uL of Detection Reagent A working solution to each well Incubate for 1 hour at 37 C after covering it with the Plate sealer Aspirate the solution and wash with 350uL of 1x Wash Solution to each well using a squirt bottle multi channel pipette manifold dispens
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6. 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 TMB Substrate is easily contaminated Please protect it from light 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 BRCA2 Standards or samples are then added to the appropriate microtiter plate wells with a biotin conjugated antibody specific to BRCAZ2 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 BRCA2 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 BRCA2 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
7. dards 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 10uL for once pipetting The reconstituted Standards Detection Reagent A and Detection Reagent B can be used only once If crystals have formed in the Wash Solution concentrate 30x 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 ie We are 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 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 0 01mol L PBS PH 7 0 7 2 If the samples are not indicated in the manual a preliminary experiment to determine the validity of the kit is necessary Tissue or cell extraction samples prepared by chemical lysis buffer may cause unexpected ELISA results due to the impacts from certain chemicals 1304 Langham Creck Dr Suite 2
8. er 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 Add 100uL of Detection Reagent B working solution to each well Incubate for 30 minutes at 37 C after covering it with the Plate sealer Repeat the aspiration wash process for total 5 times as conducted in step 4 Add 90uL of Substrate Solution to each well Cover with a new Plate sealer Incubate for 15 25 minutes at 37 C Don t exceed 30 minutes Protect from light The liquid will turn blue by the addition of Substrate Solution 8 Add 50uL 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 Note 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 20 C 2 Samples or reagents addition Please use the freshly prepared Standard Please carefully add sa
9. iluent and 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 Ong mL 1304 Langham Creck Dr Suite 226 Houston TX TTR USA 00 960 7402 www choud clhone us mailar chvud cloneus Export Processing fone Wuhan Hubei 430056 PRC 00 6 800 880 4087 www clowd clone com mail cloud clonc com gt Cloud Clone Corp 500pL 500pL 500pL 500pL 500pL 500pL MNA NA NA N o a 1 Smt A Stock see Standard T i Tube 1 2 3 4 5 6 7 8 ng mL 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 to the working concentration with Assay Diluent A and B respectively 1 100 4 Wash Solution Dilute 20mL of Wash Solution concentrate 30x with 580mL of deionized or distilled water to prepare 600mL of Wash Solution 1x 5 TMB substrate Aspirate the needed dosage of the solution with sterilized tips and do not dump the residual solution into the vial again Note 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 37 C directly 3 Please carefully reconstitute Stan
10. ity Typical Standard Curve for BRCA2 Human ELISA 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 BRCA2 is typically less than 0 055ng 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 BRCA2 No significant cross reactivity or interference between BRCA2 and analogues was observed Note Limited by current skills and knowledge it is impossible for us to complete the cross reactivity detection between BRCA2 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 BRCA2 were tested 20 times on one plate respectively Inter assay Precision Precision between assays 3 samples with low middle and high level BRCA2 were tested on 3 different plates 8 replicates in each plate CV SD meanxX100 Intra Assay CV lt 10 Inter Assay CV lt 12 1304 Langham Creck Dr Suite 226 Houston
11. mples 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 1304 Langham Creck Dr Suite 226 Houston TX T7084 USA 00 RS T402 weew cloud clone os maila choud clone us Export Processing fone Wuhan Hubei 430056 PRC 00 6 800 880 4087 www clowd clone com mail cloud clonc com gt Cloud Clone Corp 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
12. 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 1304 Langham Creck Dr Suite 226 Houston TX T7084 USA 001 4496 7402 weew cloud clone os maila choud clone us Export Processing fone Wuhan Hubei 430056 PRC 00 6 800 880 4087 www clowd clone com mail cloud clonc com CD Cloud Clone Corp Note 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 5000xg Remove the supernate and assay immediately or aliquot and store at lt 20 C
13. s 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 1304 Langham Creck Dr Suite 226 Houston TX TTR USA 00 960 7402 weew clood clone os mailar chvud cloneus Export Processing fone Wuhan Hubei 430056 PRC 00 6 800 880 0087 www clowd clone com mail clowd clonc com gt Cloud Clone Corp 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 Standard Curve oe 1304 Langham Creck Dr Suite 226 Houston TX TTO84 USA 00 R96 T402 weew clood clone os mailia choud clone us Export Processing fone Wuhan Hubei 430056 PRC 00 6 800 880 4087 www clowd clone com m
14. sks to use the kit 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 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 Do not mix or substitute reagents from one kit lot to another Use only the reagents supplied by manufacturer 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 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 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 Even the same operator might get different result
15. 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 BRCA2 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 1304 Langham Creck Dr Suite 226 Houston TX TTO84 USA 00 R96 T402 weew clood clone os mailia choud clone us Export Processing fone Wuhan Hubei 430056 PRC 00 6 800 880 4087 www clowd clone com mail cloud clonc com gt Cloud Clone Corp T t o O o 12 10 oN Q0 OW 0 0 5 4 4 5 2 2 5 3 Optical Dens

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