User Manual - RayBiotech, Inc.
Contents
1. a TMB One Step Substrate 12 ml of 3 3 5 5 tetramethylbenzidine TMB i Reagent Item H buffer solution Stop Solution Item 1 8 ml of 0 2 M sulfuric acid aaa Assay Diluent D Item K 15 ml of 5X concentrated buffer 1 month at 4 C Assay Diluent B Item E 15 ml of 5X concentrated buffer 1 month at 4 C Return unused wells to the pouch containing desiccant pack reseal along entire edge 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 lt ONONROND V REAGENT PREPARATION 1 Bring all reagents and samples to room temperature 18 25 C before use 2 Assay Diluent D Item K and Assay Diluent B Item E should be diluted 5 fold with deionized or distilled water before use 3 Sample dilution 1X Assay Diluent D Item K should be used for dilution of serum plasma and cell culture supernatant samples The suggested dilution for normal serum plasma is 1 000 fold For example add 2 ul of serum or plasma into a tube with 38 ul 1X Assay Diluent D to prepare a 20 fold diluted sample Mix thoroughly and then pipette 5 ul of prepared 20 fold dil2. 7 Ralpha Kremen 1 Limitin Lipocalin 2 LOX 1 Marapsin MBL 2 Meteorin Nope NOV Osteoactivin OX40 Ligand P Cadherin Periostin PIGF 2 Progranulin Prostasin Renin 1 Testican 3 TIM 1 TRAIL Tryptase epsilon X TROUBLESHOOTING GUIDE Inaccurate pipetting Improper standard dilution Improper preparation of standard and or biotinylated antibody Too brief incubation times Inadequate reagent volumes or improper dilution Low signal e Inaccurate pipetting Air bubbles in wells Plate is insufficiently washed Contaminated wash buffer e Improper storage of the ELISA kit e Stop solution Low sensitivity e Check pipettes Briefly centrifuge Item C and dissolve the powder thoroughly by gently mixing Briefly spin down vials before opening Dissolve the powder thoroughly Ensure sufficient incubation time assay procedure step 2 may be done overnight Check pipettes and ensure correct preparation Check pipettes Remove bubbles in wells Review the manual for proper wash If using a plate washer ensure that all ports are unobstructed Make fresh wash buffer Store your standard at lt 70 C after reconstitution others at 4 C Keep substrate solution protected from light e Add stop solution to each well before reading plate RayBio ELISA Kits Over 2 000 ELISA kits available visit www RayBiotech com ELISA Kits html for details This product is for research use only 2015 RayBiot
3. RayBio Mouse Fetuin A ELISA Kit Catalog ELM FetuinA User Manual Last revised December 7 2015 Caution Extraordinarily useful information enclosed Ke RayBiotech The protein array pioneer ISO 13485 Certified 3607 Parkway Lane Suite 100 Norcross GA 30092 Tel 1 888 494 8555 Toll Free or 770 729 2992 Fax 770 206 2393 Web www RayBiotech com Email info raybiotech com RayBiotech Inc RayBio Mouse Fetuin A ELISA Kit Protocol Table of Contents Pose invoamion Cd sensed essere Cd V Additional Materials Required Reagent Preparation VI Assay Procedure VII Assay Procedure Summary Calculation of Results A Typical Data B Sensitivity EEE C Spiking amp Recovery D Linearity E Reproducibility Specificity Troubleshooting Guide 11 0 WMO N on A A TB ol o ae Please read the entire manual carefully before starting your experiment Il INTRODUCTION The RayBio Mouse Fetuin A ELISA kit is an in vitro enzyme linked immunosorbent assay for the quantitative measurement of mouse Fetuin A in serum plasma collect plasma using EDTA as an anticoagulant Heparin and Citrate are not recommended for use in this assay and cell culture supernatants This assay employs an antibody specific for mouse Fetuin A coated on a 96 well plate Standards and samples are pipetted into the wells and Fetuin A present in a sample is bound to the wells by the immobilized anti
4. body The wells are washed and biotinylated anti mouse Fetuin A antibody is added After washing away unbound biotinylated antibody HRP conjugated streptavidin is pipetted to the wells The wells are again washed a TMB substrate solution is added to the wells and color develops in proportion to the amount of Fetuin A bound The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm ll STORAGE The entire kit may be stored at 20 C for up to 1 year from the date of shipment Avoid repeated freeze thaw cycles The kit may be stored at 4 C for up to 6 months For extended storage it is recommended to store at 80 C For prepared reagent storage see table below lil REAGENTS Size Description Storage Stay Stability Fetuin A Feun A Micropiate tem A Item A Feun AMicropiate tema wells 12 Ce x 8 wells coated with anti imonha month at aor Mouse Eli A Ween Puror Seer 25 ml 25 mi of 20x concentrated soon 20X concentrated solution 1 month at ft monthata Seer Item B Standard Protein temo Standard Protein temo Item C 2 vials of Mouse Fetuin A 1 vial is oe et to run 1 jt wookat 20 at jt wookat 20 each standard in oe et Detection Fe aa aan Fetuin A 2 vials of oer anti Mouse Fetuin A Each 5 So 5 Sdaysata at 4 Sdaysata A F vial is oer to assay half the microplate HRP ee 200 ao eee 200X concentrated HRP conjugated a not store and Concentrate ee G ao eee
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6. hould be diluted 80 fold with 1X Assay Diluent B Item E and used in step 5 of Part VI Assay Procedure Briefly spin the HRP Streptavidin concentrate vial Item G and pipette up and down to mix gently before use as precipitates may form during storage 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 do not store the diluted solution for next day use Mix well VI ASSAY PROCEDURE 1 2 3 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 Label removable 8 well strips as appropriate for your experiment Add 100 ul of each standard see Reagent Preparation step 3 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 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 to
7. ot 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 D 450 nm 0 1 4 OD 0 01 tT T T T 1 1 10 100 1000 10000 Mouse Fetuin A concentration ng ml B SENSITIVITY The minimum detectable dose of Mouse Fetuin A was determined to be 8 ng ml Minimum detectable dose is defined as the analyte concentration resulting in an absorbance that is 2 standard deviations higher than that of the blank diluent buffer C SPIKING amp RECOVERY Recovery was determined by spiking various levels of Mouse php echo Fetuin A into the sample types listed below Mean recoveries are as follows Sample Type Average Recovery Range Serum 93 79 82 104 Plasma 86 27 75 99 Cell culture media 78 01 68 90 D LINEARITY Sample Type Serum Plasma Cell Culture Media 1 2 Average of Expected 85 22 82 63 102 7 Range 78 93 76 87 97 108 1 4 Average of Expected 74 40 75 41 77 89 Range 69 79 69 81 70 86 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 Mouse B7 1 BAFF R BTC C5a CCL6 CD48 CD6 Chemerin Clusterin CXCL15 Cystatin C DAN DLL4 EDAR Endocan H60 IL 33 IL
8. uted sample into a tube with 245 ul 1X Assay Diluent D to prepare a final 1 000 fold diluted sample Note Levels of Fetuin A may vary between different samples Optimal dilution factors for each sample must be determined by the investigator 4 Preparation of standard Briefly spin a vial of Item C Add 400 ul of 1X Assay Diluent D Item K Assay Diluent D should be diluted 5 fold with deionized or distilled water before use into Item C vial to prepare a 2 000 ng ml standard Dissolve the powder thoroughly by a gentle mix Pipette 300 ul of 1X Assay Diluent D into each tube Use the stock standard solution to produce a dilution series shown below Mix each tube thoroughly before the next transfer 1X Assay Diluent D serves as the zero standard 0 ng ml Standard Item C 200u1 200ul 200l 200n 200ul 200u E H H p H H H AANAND BRERBHE 2 000 800 320 128 51 20 20 48 8 192 0 ngml ngml ngml ngm ngm ngml ng ml ng 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 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 s
9. wels 5 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 6 Discard the solution Repeat the wash as in step 4 7 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 8 Discard the solution Repeat the wash as in step 4 9 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 10 Add 50 ul of Stop Solution Item I to each well Read at 450 nm immediately V I 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 Vill 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 pl
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