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Human TACE ELISA Kit(KT20362) User Manual

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1. Human TACE ELISA Kit KT20362 User Manual For research use only Not intended for diagnostic testing TD Won rape ASABGENT II III IV VI VII VIII xe HOO Wp TABLE OF CONTENTS Introduction esses 2 Reagents tet eo Eran His 2 Mir cao 3 Additional Materials Required 3 Reagent Preparation ssessss 3 Assay Procedure csssssssssssseeene 5 Assay Procedure Summary 7 Calculation of Results Typical Data nepie eroien krere 8 Sensitivity iiie a a a aka 8 RECOVERY usce T ENE sts ANNEES 8 Lanearity i iiu eese em eS EE ey pee eges 9 Reproducibility c cc ccecceceeeene ences enens 9 SpecitiCIty uii see edere NE ERR ARE MEE ERR 9 Troubleshooting Guide 10 I INTRODUCTION The Human TACE TNF alpha converting enzyme ELISA Enzyme Linked Immunosorbent Assay kit is an in vitro enzyme linked immunosorbent assay for the quantitative measurement of human TACE in serum plasma cell culture supernatants and urine This assay employs an antibody specific for human TACE coated on a 96 well plate Standards and samples are pipetted into the wells and TACE present in a sample is bound to the wells by the immobilized antibody The wells are washed and biotinylated anti human TACE antibody is added After washing away unbound biotinylated antibody HRP conjugated streptavidin is pipetted to the wells The wells are a
2. pack reseal along entire edge Note the kit can be used within one year if the whole kit is stored at 20 C Avoid repeated freeze thaw cycles IV ADDITIONAL MATERIALS REQUIRED Microplate reader capable of measuring absorbance at 450 nm Precision pipettes to deliver 2 ul to 1 ml volumes Adjustable 1 25 ml pipettes for reagent preparation 100 ml and 1 liter graduated cylinders Absorbent paper Distilled or deionized water Log log graph paper or computer and software for ELISA data analysis Tubes to prepare standard or sample dilutions OAYDMABRWN V REAGENT PREPARATION 1 Bring all reagents and samples to room temperature 18 25 C before use 2 Sample dilution If your samples need to be diluted Assay Diluent C Item L should be used for dilution of serum plasma culture supernatants urine Suggested dilution for normal serum plasma 2 fold Please note that levels of the target protein may vary between different specimens Optimal dilution factors for each sample must be determined by the investigator 3 Assay Diluent B Item E should be diluted 5 fold with deionized or distilled water before use 4 Preparation of standard Briefly spin the vial of Item C Add 400 ul Assay Diluent C Item L into Item C vial to prepare a 50 ng ml standard solution Dissolve the powder thoroughly by a gentle mix Add 50 ul TACE standard from the vial of Item C into a tube with 450 ul Assay Diluent C to prepar
3. Inaccurate pipetting 1 Check pipettes 4 High background 1 Plate is insufficiently 1 Review the manual washed for proper wash If using an a plate washer check that all ports are unobstructed 2 Contaminated wash 2 Make fresh wash buffer buffer 5 Low sensitivity 1 Improper storage of the ELISA kit 2 Stop solution Store your standard at lt 20 C after reconstitution others at 4 C Keep substrate solution protected from light Stop solution should be added to each well before measure Note This product is for research use only ag a USA Abgent Inc Toll Free 888 735 7227 Or 858 875 1900 info_us abgent wuxiapptec com CHINA Abgent Suzhou 86 512 69369088 sales abgent wuxiapptec com EUROPE Abgent Europe 44 0 1235 854042 eurosales abgent wuxiapptec com For other countries www abgent com
4. ample Type Average Recovery Range 96 Serum 112 3 85 132 Plasma 101 1 72 128 Cell culture media 98 65 89 124 D LINEARITY Sample Type Serum Plasma Cell Culture Media 1 2 Average of Expected 84 82 85 56 119 4 Range 96 84 103 76 93 87 132 1 4 Average of Expected 76 87 77 54 76 39 Range 96 69 85 68 91 67 85 E REPRODUCIBILITY Intra Assay CV lt 10 Inter Assay CV lt 12 IX SPECIFICITY Cross Reactivity This ELISA kit shows no cross reactivity with the following cytokines tested human Angiogenin BDNF BLC CNTF ENA 78 FGF 4 IL la IL 1B IL 2 IL 3 IL 4 IL 5 IL 6 IL 7 IL 8 IL 9 IL 11 IL 12 p70 IL 12 p40 IL 13 IL 15 IL 309 IP 10 FGF 4 FGF 6 FGF 7 G CSF GDNF GM CSF IFN y IGFBP 2 IGF BP 3 IGF BP 4 Leptin OB MCP 1 MCP 2 MCP 3 MDC MIF MIG MIP la MIP 1 B MIP 16 PARC PDGF RANTES SCF SDF 1alpha TARC TGF B TIMP 1 TIMP 2 TNF a TNF p TPO VEGF X TROUBLESHOOTING GUIDE Problem Cause Solution 1 Poor standard 1 Inaccurate pipetting 1 Check pipettes curve 2 Improper standard 2 Ensure briefly spin dilution the vial of Item C and dissolve the powder thoroughly by a gentle mix 2 Low signal 1 Too brief incubation times Ensure sufficient incubation time assay procedure step 2 change to over night 2 Inadequate reagent 2 Check pipettes and volumes or improper ensure correct dilution preparation 3 Large CV 1
5. diately VII ASSAY PROCEDURE SUMMARY 1 Prepare all reagents samples and standards as instructed 2 Add 100 ul standard or sample to each well Incubate 2 5 hours at room temperature or over night at 4 C 3 Add 100 ul prepared biotin antibody to each well Incubate 1 hour at room temperature 4 Add 100 ul prepared Streptavidin solution Incubate 45 minutes at room temperature 5 Add 100 ul TMB One Step Substrate Reagent to each well Incubate 30 minutes at room temperature 6 Add 50 ul Stop Solution to each well Read at 450 nm immediately VIII CALCULATION OF RESULTS Calculate the mean absorbance for each set of duplicate standards controls and samples and subtract the average zero standard optical density Plot the standard curve on log log graph paper or using Sigma plot software with standard concentration on the x axis and absorbance on the y axis Draw the best fit straight line through the standard points A TYPICAL DATA These standard curves are for demonstration only A standard curve must be run with each assay Assay Diluent C OD 450 nm eo 0 01 10 100 1000 10000 Human TACE concentration pg ml B SENSITIVITY The minimum detectable dose of TACE is typically less than 70 pg ml C RECOVERY Recovery was determined by spiking various levels of TACE into normal human serum plasma and cell culture media Mean recoveries are as follows S
6. don t store the diluted solution for next day use Mix well VI ASSAY PROCEDURE 1 Bring all reagents and samples to room temperature 18 25 C before use It is recommended that all standards and samples be run at least in duplicate 2 Add 100 pl of each standard see Reagent Preparation step 2 and sample into appropriate wells Cover well and incubate for 2 5 hours at room temperature or over night at 4 C with gentle shaking 3 Discard the solution and wash 4 times with 1x Wash Solution Wash by filling each well with Wash Buffer 300 ul using a multi channel Pipette or autowasher Complete removal of liquid at each step is essential to good performance After the last wash remove any remaining Wash Buffer by aspirating or decanting Invert the plate and blot it against clean paper towels Add 100 ul of 1x prepared biotinylated antibody Reagent Preparation step 6 to each well Incubate for 1 hour at room temperature with gentle shaking Discard the solution Repeat the wash as in step 3 Add 100 ul of prepared Streptavidin solution see Reagent Preparation step 7 to each well Incubate for 45 minutes at room temperature with gentle shaking Discard the solution Repeat the wash as in step 3 Add 100 ul of TMB One Step Substrate Reagent Item H to each well Incubate for 30 minutes at room temperature in the dark with gentle shaking Add 50 ul of Stop Solution Item I to each well Read at 450 nm imme
7. e a 5 000 pg ml standard solution Pipette 30011 Assay Diluent C into each tube Use the stock standard solution to produce a dilution series shown below Mix each tube thoroughly before the next transfer Assay Diluent C serves as the zero standard 0 pg ml 50 ul standard 300 ul 450 ul 300u 300g 300g 3001 3004 u 5 000 2 500 1 250 625 312 5 156 3 78 15 0 pg ml pg ml pg ml pg ml pg ml pg ml pg ml pg ml 5 If the Wash Concentrate 20x Item B contains visible crystals warm to room temperature and mix gently until dissolved Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to yield 400 ml of 1x Wash Buffer 6 Briefly spin the Detection Antibody vial Item F before use Add 100 ul of 1x Assay Diluent B Item E into the vial to prepare a detection antibody concentrate Pipette up and down to mix gently the concentrate can be stored at 4 C for 5 days The detection antibody concentrate should be diluted 80 fold with 1x Assay Diluent B and used in step 4 of Part VI Assay Procedure 7 Briefly spin the HRP Streptavidin concentrate vial Item G and pipette up and down to mix gently before use HRP Streptavidin concentrate should be diluted 200 fold with 1x Assay Diluent B Item E For example Briefly spin the vial Item G and pipette up and down to mix gently Add 50 ul of HRP Streptavidin concentrate into a tube with 10 ml 1x Assay Diluent B to prepare a 200 fold diluted HRP Streptavidin solution
8. gain washed a TMB substrate solution is added to the wells and color develops in proportion to the amount of TACE bound The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm Il REAGENTS 1 TACE Microplate Item A 96 wells 12 strips x 8 wells coated with anti human TACE 2 Wash Buffer Concentrate 20x Item B 25 ml of 20x concentrated solution Standards Item C 2 vials recombinant human TACE 4 Assay Diluent C Item L 30 ml of diluent buffer For Standard Sample serum plasma samples cell culture medium urine diluent 5 Assay Diluent B Item E 15 ml of 5x concentrated buffer For detection antibody and HRP Streptavidin diluent 6 Detection Antibody TACE Item F 2 vial of biotinylated anti human TACE each vial is enough to assay half microplate 7 HRP Streptavidin concentrate Item G 200 ul 200x concentrated HRP conjugated streptavidin 8 TMB One Step Substrate Reagent Item H 12 ml of 3 3 5 5 tetramethylbenzidine TMB in buffered solution 9 Stop Solution Item I 8 ml of 0 2 M sulfuric acid w Ill STORAGE May be stored for up to 6 months at 2 to 8 C from the date of shipment Standard recombinant protein should be stored at 20 C or 80 C recommended at 80 C after reconstitution Opened Microplate Wells or reagents may be stored for up to 1 month at 2 to 8 C Return unused wells to the pouch containing desiccant

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