Manual - RayBiotech, Inc.
Contents
1. 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 Item E2 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 400 fold with 1X Assay Diluent Item E2 For example Briefly spin the vial Item G and pipette up and down to mix gently Add 30 ul of HRP Streptavidin concentrate into a tube with 12 ml 1X Assay Diluent to prepare a 400 fold diluted HRP Streptavidin solution don t 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 performance2. After the last wash remove any remaining Wash Buffer by aspirating or decanting Invert the plate and blot it against clean paper towels 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 a
3. RayBio Rat IL 6 ELISA Kit For Lysates Catalog ELR IL6 CL 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 1 RayBiotech Inc RayBio Rat IL 6 ELISA Kit For Lysates Protocol Table of Contents Pose Invoanion E sure essen v roor maras Reged OOOO Tione OOOO Pere SS assay Pace sonnen OOO OO Calculation of Results A Typical Data B Sensitivity FEET V l VI C Spiking amp Recovery D Linearity E Reproducibility Specificity Troubleshooting Guide 11 F FF 0 WMO N on A A TB ol o Please read the entire manual carefully before starting your experiment Il INTRODUCTION The RayBio Rat IL 6 ELSA Enzyme Linked Immunosorbent Assay kit is an in vitro enzyme linked immunosorbent assay for the quantitative measurement of Rat IL 6 cell lysate and tissue lysate This assay employs an antibody specific for Rat IL 6 coated on a 96 well plate Standards and samples are pipetted into the wells and IL 6 present in a sample is bound to the wells by the immobilized antibody The wells are washed and biotinylated anti Rat IL 6 antibody is added After washing away unbound biotinylated antibody HRP c
4. f original lysate solution though more concentrated is better We also recommend the addition of protease inhibitors not included to the lysis buffer prior to use Detailed recommendations on lysis preparation may be found here www raybiotech com tips on sample preparation html Note Levels of IL 6 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 500 ul 1X Sample Diluent Buffer Item D2 into Item C vial to prepare a 10 000 pg ml standard solution Dissolve the powder thoroughly by a gentle mix Pipette 300 pl 1X Sample Diluent Buffer into each tube Use the 10 000 pg ml standard solution to produce a dilution series shown below Mix each tube thoroughly before the next transfer 1X Sample Diluent Buffer serves as the zero standard 0 pg ml Standard Item C 500 ul GIS IS OS OS A 200ul 200 2001 200ul 200m 200 JBE 10 000 4 000 1 600 640 256 102 4 40 96 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 Briefly spin the Detection Antibody vial Item F before use Add 100 ul of 1X Assay Diluent Item E2 into the vial to prepare a detection antibody
5. nd 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 Sample Diluent Buffer 450 nm 0 1 4 OD 0 01 j r i 10 100 1 000 10 000 Rat IL 6 concentration pg ml B SENSITIVITY The minimum detectable dose of Rat IL 6 was determined to be 15 pg 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 Rat php echo IL 6 into the sample types listed below Mean recoveries are as follows Sample Type Average Recovery Range Tissue lysate 79 57 70 87 Cell lysate 75 67 67 85 D LINEARITY Sample Type Tissue Cell Lysate lysate 1 2 Average of 116 2 132 2 Expected Range 105 124 121 142 1 4 Average of 77 88 131 1 Expected Range 68 86 120 142 E REPRODUCIBILITY Intra Assay CV lt 10 Inter Assay CV lt 12 IX SPECIFICITY The antibody pair provided in this kit recognizes rat IL 6 X TROUBLESHOOTING GUIDE Inaccurate pipetting Improper standard dilution Improper
6. onjugated 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 IL 6 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 Saoi Stability IL 6 Microplate Item A 12 strips x 8 wells coated with anti Rat imanna month at aor Ween Burer eer 25 ml 25 mi of 20x concentrated soon 20X concentrated solution 1 month at month ata eer Item B Standard Protein temo Standard Protein temo Item C 2 vials of Rat IL 6 1 vial is oe eet to run each 1 jt wockat 30 at jt wockat 30 standard in oe eet oe Peele eau IL 6 Item 2 vials of er en aa anti Rat IL 6 Each vial is 5 5 Bosaso at 4 Bosaso e a to assay half the microplate HRP T T 200 m ee 400X concentrated HRP conjugated a not store and Concentrate ee G m ee a TMB One Step Substrate 12 ml of 3 3 5 5 tetramethylbenzidine TMB i eae Item H buffer solution Stop Solution Item Solution Item l 8 ml of 0 2 M sulfuric acid aaa Bufer hemi 10 10 m1 of 8X concent
7. 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 RayBiotech Inc
8. rated buer 10 m1 of 8X concentrated buer 5X concentrated buffer 1 Imoma at Imoma Assay Diluent tem E2 Diluent Item Assay Diluent tem E2 15 15 mI of 5X concentrated buffer 15 mI of 5X concentrated buffer 5X concentrated buffer 1 1 month at4 C at 1 month at4 C Cell lysate buffer Item J 5 ml of 2X cell lysate 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 4 lt ONONROND V REAGENT PREPARATION 1 Bring all reagents and samples to room temperature 18 25 C before use 2 Sample Diluent Buffer Item D2 and Assay Diluent Item E2 should be diluted 5 fold with deionized or distilled water before use Cell lysate buffer Item J should be diluted 2 fold with deionized or distilled water for cell lysate and tissue lysate 3 Sample dilution Tissue lysate and cell lysate samples should be diluted at least 5 fold with 1X Sample Diluent Buffer Item D2 Generally we recommend a minimum of 1 mg of protein per 1 ml o
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