RayBio® Human MMP-13 ELISA Kit
Contents
1. Human MMP 13 ELISA Kit Protocol 5 VI ASSAY PROCEDURE I 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 Add 100 ul 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 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 RayBio Human MMP 13 ELISA Kit Protocol 6 9 Add 50 ul of Stop Solution Item I to e2. into human serum plasma and cell culture media Mean recoveries are as follows Sample Type Average Recovery Range Serum 91 99 82 102 Plasma 93 13 83 103 Cell culture media 94 72 84 103 RayBio Human MMP 13 ELISA Kit Protocol 8 D LINEARITY Sample Type Serum Plasma Cell Culture Media 1 2 Average 96 of Expected 92 93 94 Range 82 103 83 104 82 103 1 4 Average of Expected 90 92 93 Range 83 102 84 103 83 102 E REPRODUCIBILITY Intra Assay CV lt 10 Inter Assay CV lt 12 IX SPECIFICITY Cross Reactivity This ELISA kit shows no cross reactivity with any of the cytokines tested e g Angiopoietin 1 Angiostatin B7 1 BMP 7 CD14 CD30 CD40 CD40 Ligand CTLA 4 CXCL16 Dkk 4 DR6 Endostatin E Selectin Follistatin HB EGF HV EM ICAM 2 IGF II IL 10 Ra IL 10 Rb IL 18 IL 9 IL 2 Ra IL 2 Rb IL 5 Ra LAP L Selectin M CSF R MMP 1 2 3 7 8 9 10 and 12 PDGF AB SDF 1b Tie 1 Tie 2 TIMP 3 RayBio Human MMP 13 ELISA Kit Protocol 9 X REFERENCES 1 Borkakoti N Matrix metalloproteases variations on a theme Progress in Biophysics and Molecular Biology 70 1 73 94 1998 2 Giambernardi TA et al Overview of matrix metalloproteinase expression in cultured human cells Matrix Biology 16 8 483 496 1998 3 Nagase H Activation mechanisms of matrix metalloproteinases Biol Chem 378 3 4 151 160 1997 RayBio Human MMP 13 ELISA Kit Protocol 10 XI TROUBLESHOOTING GUI
3. 3 bound The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm RayBio Human MMP 13 ELISA Kit Protocol 2 II REAGENTS 1 MMP 13 Microplate Item A 96 wells 12 strips x 8 wells coated with anti human MMP 13 2 Wash Buffer Concentrate 20x Item B 25 ml of 20x concentrated solution 3 Standards Item C 2 vials recombinant human MMP 13 4 Assay Diluent Item E 15 ml of 5x concentrated buffered For Standard Sample serum plasma samples cell culture medium urine diluent 5 Detection Antibody MMP 13 Item F 2 vial of biotinylated anti human MMP 13 each vial is enough to assay half microplate 6 HRP Streptavidin Concentrate Item G 200 ul of 700x concentrated HRP conjugated streptavidin 7 TMB One Step Substrate Reagent Item H 12 ml of 3 3 5 5 tetramethylbenzidine TMB in buffered solution 8 Stop Solution Item I 8 ml of 0 2 M sulfuric acid HI 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 store for up to 1 month at 2 to 8 C Return unused wells to the pouch containing desiccant 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 MA
4. Assay Diluent 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 serves as the zero standard 0 pg ml RayBio Human MMP 13 ELISA Kit Protocol 4 80 ul standard 586 7 ul 2000 200 0 200g 200g 2004 2004 6000 2000 666 7 2222 74 07 24 69 8 23 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 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 and used in step 4 of Part VI Assay Procedure 7 Briefly spin the HRP Streptavidin concentrate vial Item G before use HRP Streptavidin concentrate should be diluted 700 fold with 1x Assay Diluent 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 14 ml Ix Assay Diluent to prepare a 700 fold diluted HRP Streptavidin solution don t store the diluted solution for next day use Mix well RayBio
5. DE Problem Cause Solution 1 Poor standard curve 1 Inaccurate pipetting 2 Improper standard dilution 1 Check pipettes 2 Ensure a brief spin of Item C and dissolve the powder thoroughly by a gentle mix 2 Low signal 1 Too brief incubation 1 Ensure sufficient times incubation time assay procedure step 2 may change to over night 2 Inadequate reagent 2 Check pipettes and volumes or improper ensure correct dilution preparation 3 Large CV 1 Inaccurate pipetting 1 Check pipettes 4 High background 1 Plate is insufficiently washed 2 Contaminated wash buffer 1 Review the manual for proper wash If using a plate washer check that all ports are unobstructed 2 Make fresh wash buffer 5 Low sensitivity 1 Improper storage of the ELISA kit 2 Stop solution 1 Store your standard at 20 C after reconstitution others at 4 C Keep substrate solution protected from light 2 Stop solution should be added to each well before measure RayBio Human MMP 13 ELISA Kit Protocol 11 RayBio ELISA kits Over 200 ELISA kits custom ELISA kit choose from over 500 list visit www raybiotech com for details RayBio Human MMP 13 ELISA Kit Protocol 12 This product is for research use only 2004 RayBiotech Inc RayBio Human MMP 13 ELISA Kit Protocol 13
6. RayBio Human MMP 13 ELISA Kit User Manual Revised Mar 1 2012 RayBio Human MMP 13 ELISA Kit Protocol Cat ELH MMP13 001 RayBiotech Inc We Provide You With Excellent Protein Array System And Service Tel Toll Free 1 888 494 8555 or 770 729 2992 Fax 770 206 2393 Web www raybiotech com Email info raybiotech com II M IV VI VII VII H Uns XI RayBiotech Inc RayBio Human MMP 13 ELISA Kit Protocol TABLE OF CONTENTS Introd DO sedo Due ad hPa is Paare OM OH odo oo REUS PERPE CUM nRMMdM OUS rng Additional Materials Required Reagent Preparation ssuse Assay Procedure cceeeeeeeeeeees Assay Procedure Summary Calculation of Results Typical Dbdusesesesencscuenoo extre y eee ERR SSMSIUIVILV PPP ROCOVOEV 2210 casero PARERE URARSATERSDEMERIG UN PCAN V s TP Reproducibility cee ceee eee eee DPE CILIC lb oobersxetosevuesatun vuU ereer errre Troubleshooting Guide Reference eeeeeee nennen RayBio Human MMP 13 ELISA Kit Protocol 1 I INTRODUCTION Matrix metalloproteinases MMPs are a family of zinc dependent endopeptidases that degrade extracellular matrix proteins MMPs have been linked with a wide array of biological activities and play important roles during organ development and pathological processes Collectively MMPs are key enzymes fo
7. TERIALS REQUIRED Microplate reader capable of measuring absorbance at 450 nm 2 Precision pipettes to deliver 2 ul to 1 ml volumes 3 Adjustable 1 25 ml pipettes for reagent preparation 4 100 ml and 1 liter graduated cylinders RayBio Human MMP 13 ELISA Kit Protocol 3 5 Absorbent paper Distilled or deionized water 7 Log log graph paper or computer and software for ELISA data analysis 8 Tubes to prepare standard or sample dilutions ON 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 1x Assay Diluent Item E 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 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 and then add 400 ul 1x Assay Diluent Item E into Item C vial to prepare a 50 ng ml standard Dissolve the powder thoroughly by a gentle mix Add 80 ul MMP 13 standard from the vial of item C into a tube with 586 7 ul 1x Assay Diluent Buffer for serum plasma samples cell culture medium urine to prepare a 6000 pg ml stock standard solution Pipette 400ul 1x
8. ach well Read at 450 nm immediately VII ASSAY PROCEDURE SUMMARY 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 RayBio Human MMP 13 ELISA Kit Protocol 7 A TYPICAL DATA These standard curves are for demonstration only A standard curve must be run with each assay Assay Diluent Buffer OD 450 nm 0 1 4 0 01 r 1 10 100 1000 10000 Human MMP 13 concentration pg ml B SENSITIVITY The minimum detectable dose of MMP 13 is typically less than 6 pg ml C RECOVERY Recovery was determined by spiking various levels of haman MMP 13
9. r the metabolism of extracellular matrix proteins including fibrillar and non fibrillar collagens fibronectin laminin and basement membrane or interstitial stroma glycoproteins Under physiological conditions MMPs are involved in extracellular degradation and breakdown of matrix proteins during normal tissue remodelling processes such as wound healing pregnancy and angiogenesis MMP 13 is expressed by stromal cells in breast carcinomas and produced at significant levels during fetal ossification and in arthritic processes The RayBio Human MMP 13 ELISA Enzyme Linked Immunosorbent Assay kit is an in vitro enzyme linked immunosorbent assay for the quantitative measurement of human MMP 13 in serum human MMP 13 concentration is pretty low in normal serum plasma it may not be detected in this assay plasma Collect plasma using heparin as an anticoagulant EDTA and Citrate are not recommended cell culture supernatants and urine This assay employs an antibody specific for human MMP 13 coated on a 96 well plate Standards and samples are pipetted into the wells and MMP 13 present in a sample is bound to the wells by the immobilized antibody The wells are washed and biotinylated anti human MMP 13 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 MMP 1
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