User Manual - RayBiotech, Inc.
Contents
1. 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 Inc2. isoforms are expressed in a more limited spectrum of cells and tissues It is normally secreted as an inactive or latent complex The RayBio Human TGF beta1 ELISA Enzyme Linked mmunosorbent Assay kit is an in vitro enzyme linked immunosorbent assay for the quantitative measurement of human TGF beta in serum plasma and cell culture supernatants This assay employs an antibody specific for human TGF beta1 coated on a 96 well plate Standards and samples are pipetted into the wells and TGF beta1 present in a sample is bound to the wells by the immobilized antibody The wells are washed and biotinylated anti human TGF beta1 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 TGF beta1 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 a beta 1 Item 96 wells 12 na x 8 wells coated with anti month at aor F 1 month at 4 month at aor
3. 9 g HEPES Mix through Bring final volume to 100 mL with deionized water 2 5 N Acetic Acid 10 M Urea 250 ml Add 150 2 g of Urea into 100 mL deionized water Mix bottle until dissolved Slowly add 35 9 mL of Glacial Acetic Acid Mix through Bring final volume to 250 ml with deionized water 2 7 N NaOH 1 M HEPES 250 ml Add 67 5 ml of 10 N NaOH into 140 ml deionized water Mix bottle Add 59 5 g HEPES Mix through Bring final volume to 250 mL with deionized water TGF beta 1 SAMPLE ACTIVATION PROCEDURE To activate latent TGF beta 1 to the immunoreactive form follow the activation procedure outlined below Assay samples after neutralization pH 7 0 7 6 Use polypropylene test tubes Notes Do not activate the kit standards The kit standards contain active rhnT GF beta1 1 cell culture supernatants Add 0 1 ml 1 N HCI into 0 5 mL cell culture supernate or urine Mix tube thoroughly Incubate for 10 minutes at room temperature Neutralize the acidified sample by adding 0 1 ml 1 2 N NaOH 0 5 M HEPES PH 7 0 7 6 Mix tube thoroughly Assay immediately The activated sample may be diluted with 1X Assay Diluent B for cell culture supernatants The concentration read off the standard curve must be multiplied by the dilution factor 2 Serum plasma Add 0 1 ml 2 5 N Acetic Acid 10 M Urea to 0 1 ml serum Mix tube thoroughly Incubate for 10 minutes at room temperature Neutralize the acidified sample by adding 0 1 ml 2 7 N NaOH 1 M H
4. E ae beta 1 Wash Suet ie 25 ml 25 mi of 20x concentrated soon 20X concentrated solution 1 month at ft monthata ie Item B Standard Protein temo Standard Protein temo Item C 2 vials of Human TGF beta 1 1 vial is eo eet to 1 jt wockat 30 at jt wockat 30 run each standard in eo eet Re Fre geeaaraa TGF beta 2 vials of Be Eel eerie l anti Human TGF beta 1 5 nee 5 Sdaysata at 4 Sdaysata Fre geeaaraa F Each vial is Be Eel eerie l to assay half the microplate HRP O a 200 o N 500X concentrated HRP conjugated a not store and Concentrate O a G o N 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 anand Assay Diluent A Item D 30 ml of diluent buffer 0 09 sodium azide as preservative 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 ONOnNROND V REAGENT PREPARATI
5. EPES Mix tube thoroughly Assay immediately The activated sample may be diluted with Assay Diluent A The concentration read off the standard curve must be multiplied by the dilution factor 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 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 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 4 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 o
6. ON 1 Bring all reagents and samples to room temperature 18 25 C before use 2 Assay Diluent B Item E should be diluted 5 fold with deionized or distilled water before use 3 Sample dilution Assay Diluent A Item D should be used for dilution of serum and plasma samples 1X Assay Diluent B Item E should be used for dilution of cell culture supernatant samples The suggested dilution for normal serum plasma is 3 fold after treatment see activation steps on page 6 Note Levels of TGF beta 1 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 700 ul Assay Diluent A Item D for serum plasma or 1X Assay Diluent B Item E for cell culture supernatants to prepare a 300 ng ml standard Dissolve the powder thoroughly by a gentle mix Add 120 ul TGF beta1 standard from the vial of Item C into a tube with 480 ul Assay Diluent A or 1X Assay Diluent B to prepare a 60 ng ml stock standard solution Pipette 400 ul Assay Diluent A or 1X Assay Diluent B 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 A or 1X Assay Diluent B serves as the zero standard 0 ng ml 120 ul standard 9 480 ul 200u1 200l 200p 200ul 200n 200l TS OS OS OS 60 20 6 667 2 222 0 741 0 247 0 0823 0 ng ml ng ml ng ml n
7. RayBio Human TGF beta 1 ELISA Kit Catalog ELH TGFb1 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 Human TGF beta 1 ELISA Kit Protocol Table of Contents Pose Investor 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 Transforming Growth Factor Beta TGF beta is a stable multifunctional polypeptide growth factor TGF beta exists in at least five isoforms Known as TGF beta1 TGF beta2 TGF beta3 TGF beta4 TGF beta5 Their amino acid sequences display homologies on the order of 70 80 The various TGF beta isotypes share many biological activities and their actions on cells are qualitatively similar in most cases although there are a few examples of distinct activities TGF beta is the prevalent form and is found almost ubiquitously while the other
8. centration ng ml Human TGF beta 1 concentration ng ml B SENSITIVITY The minimum detectable dose of Human TGF beta 1 was determined to be 80 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 Human php echo TGF beta 1 into the sample types listed below Mean recoveries are as follows Sample Type Average Recovery Range Serum 94 46 82 102 Plasma 95 78 93 103 Cell culture media 97 87 85 104 D LINEARITY Sample Type Serum Plasma Cell culture media 1 2 Average of Expected 92 95 95 Range 82 103 83 104 84 104 1 4 Average of Expected 93 94 94 Range 83 105 84 105 83 104 E REPRODUCIBILITY Intra Assay CV lt 10 Inter Assay CV lt 12 IX SPECIFICITY This ELISA kit shows no cross reactivity with any of the cytokines tested Human ANG CD23 Eotaxin GCSF GM CSF GRO alpha GRO beta GRO gamma l 309 IFN gamma IL 1 alpha IL 1 beta IL 3 IL 4 IL 5 IL 6 IL 7 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
9. f 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 Vil 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 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 A Assay Diluent B 10 10 1 4 E 14 c 3 ll li Q Q O o14 O o14 0 01 T T T 0 01 r T 7 0 0 1 1 10 100 0 0 1 1 10 100 Human TGF beta 1 con
10. g ml ng ml ng ml 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 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 Item E and used in step 5 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 as precipitates may form during storage HRP Streptavidin concentrate should be diluted 500 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 20 ul of HRP Streptavidin concentrate into a tube with 10 ml 1X Assay Diluent B to prepare a 500 fold diluted HRP Streptavidin solution don t store the diluted solution for next day use Mix well Reagents to activate serum plasma and cell culture supernatant samples 1 N HCI 100 ml Slowly add 8 33 mL of 12 N HCl into 91 67 ml deionized water Mix bottle 1 2 N NaOH 0 5 M HEPES 100 ml Slowly add 12 ml of 10 N NaOH into 75 mL deionized water Mix bottle Add 11
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