Human LIMP-II ELISA Kit Protocol
Contents
1. 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 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 8 Tubes to prepare standard or sample dilutions NAD d G A ra 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 samples 1x Assay Diluent B Item E should be used for dilution of culture supernatants and urine Suggested dilution for normal serum plasma 2 fold RayBio Human LIMP II ELISA Kit Protocol 3 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 should be diluted 5 fold with deionized or distilled water before use 4 Preparation of standard B2. 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 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 final 200 fold diluted HRP Streptavidin solution 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 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 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 RayBio Human LIMP II ELISA Kit Protocol 5 4 Add 100 ul of 1x prepared biotinylated antibody Reagent Preparation step 6 to each well Incubate for 1 hour at room temperature with
3. 500 list visit www raybiotech com for details RayBio Human LIMP II ELISA Kit Protocol 11 RayBio Human LIMP II ELISA Kit Protocol This product is for research use only 2004 RayBiotech Inc RayBio Human LIMP II ELISA Kit Protocol 13
4. 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 1a MIP 1 B MIP 16 PARC PDGF RANTES SCF SDF 1alpha TARC TGF B TIMP 1 TIMP 2 TNF a TNF B TPO VEGF RayBio Human LIMP II ELISA Kit Protocol 9 X TROUBLESHOOTING GUIDE 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 LIMP II ELISA Kit Protocol 10 RayBio ELISA kits Over 200 ELISA kits custom ELISA kit choose from over
5. II 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 LIMP II bound The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm II REAGENTS 1 LIMP II Microplate Item A 96 wells 12 strips x 8 wells coated with anti human LIMP II 2 Wash Buffer Concentrate 20x Item B 25 ml of 20x concentrated solution 3 Standards Item C 2 vials of recombinant human LIMP II 4 Assay Diluent C Item L 30 ml diluent buffer For Standard Sample serum plasma diluent 5 Assay Diluent B Item E 15 ml of 5x concentrated buffer For Standard Sample cell culture medium urine diluent 6 Detection Antibody Item F 2 vial of biotinylated anti human LIMP II 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 buffer solution 9 Stop Solution Item I 8 ml of 0 2 M sulfuric acid RayBio Human LIMP II ELISA Kit Protocol 2 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
6. RayBio Human LIMP II ELISA Kit User Manual Revised Mar 1 2012 RayBio Human LIMP II ELISA Kit Protocol Cat ELH LIMPII 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 VIII MOO WwW gt e RayBiotech Inc RayBio Human LIMP II ELISA Kit Protocol TABLE OF CONTENTS E DEEN 543452552 ebbe Pies RECASENS JINE E Additional Materials Required Reagent Preparation ee Assay NEE Assay Procedure Summary Calculation of Results Typical Data sucosesesot sinet Rer REI Sa DER IL EE RECOVETY eelere QWQ j Lineafity DD hh um Reproducibility E n en E Troubleshooting CGoude RayBio Human LIMP II ELISA Kit Protocol 1 mms H I INTRODUCTION The RayBio Human LIMP II ELISA Enzyme Linked Immunosorbent Assay kit is an in vitro enzyme linked immunosorbent assay for the quantitative measurement of human LIMP II in serum plasma cell culture supernatants and urine This assay employs an antibody specific for human LIMP II coated on a 96 well plate Standards and samples are pipetted into the wells and LIMP II present in a sample is bound to the wells by the immobilized antibody The wells are washed and biotinylated anti human LIMP
7. gentle shaking 5 Discard the solution Repeat the wash as in step 3 6 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 7 Discard the solution Repeat the wash as in step 3 8 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 9 Add 50 ul of Stop Solution Item I to each 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 RayBio Human LIMP II ELISA Kit Protocol 6 5 Add 100 ul TMB One Step Substrate Reagent to each well Incubate 30 minutes at room temperature l 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
8. 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 Assay Diluent B 10 10 E e eo LO LO H 3 5 0 1 4 5 0 1 0 01 r 0 01 r i 1 10 100 1000 10000 1 10 100 1000 10000 Human LIMP II concentration pg ml Human LIMP II concentration pg ml RayBio Human LIMP II ELISA Kit Protocol 7 B SENSITIVITY The minimum detectable dose of LIMP II is typically less than 10 pg ml C RECOVERY Recovery was determined by spiking various levels of human LIMP II into human serum plasma and cell culture media Mean recoveries are as follows Sample Type Average Recovery Range Serum 83 00 74 92 Plasma 79 65 71 86 Cell culture media 95 06 74 107 D LINEARITY Sample Type Serum Plasma Cell Culture Media 1 2 Average of Expected 106 2 91 05 104 5 Range 98 115 73 103 97 112 1 4 Average 96 of Expected 107 7 86 67 78 74 Range 96 117 78 94 67 86 E REPRODUCIBILITY Intra Assay CV lt 10 Inter Assay CV lt 12 RayBio Human LIMP II ELISA Kit Protocol 8 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 10 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
9. riefly spin the vial of Item C and then add 400 ul Assay Diluent C for serum plasma samples or 1x Assay Diluent B for cell culture supernates urine into Item C vial to prepare a 50 ng ml standard Dissolve the powder thoroughly by a gentle mix Add 25 ul LIMP II standard 50 ng ml from the vial of Item C into a tube with 475 ul Assay Diluent C or 1x Assay Diluent B to prepare a 2 500 pg ml standard solution Pipette 300 ul Assay Diluent C or 1x Assay Diluent B into each tube Use the 2 500 pg ml standard solution to produce a dilution series shown below Mix each tube thoroughly before the next transfer Assay Diluent C or 1x Assay Diluent B serves as the zero standard 0 pg ml 25 ul standard 475 ul 200u 200g 200p 200g 200g 200 jil 2 500 1 000 400 160 64 25 6 10 24 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 RayBio Human LIMP II ELISA Kit Protocol 4 6 Briefly spin the Detection Antibody vial Item F before use Add 100 ul of Ix Assay Diluent B 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
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