Mouse Shh-N ELISA Kit（KT20580） User Manual
Contents
1. Mouse Shh N ELISA Kit KT20580 User Manual For research use only Not intended for diagnostic testing TD waa noptec e ARGENT II III IV VI VII VIII eumouotuvxopo TABLE OF CONTENTS Introduction esses 2 R agents i sera pr RS er od Penes 2 StOTag p 3 Additional Materials Required 3 Reagent Preparation esses 3 Assay Procedure eo Le e 5 Assay Procedure Summary 7 Calculation of Results Typical Data esses id Sensi vity golden ete tide poteet redes 8 RECOVERY c REVO E EE eie 8 LEinearity io co a 9 Reproducibility uri ensi 9 Specificity iov io eoe Yea Rue 9 Troubleshooting Guide 10 I INTRODUCTION The Mouse Shh N Sonic Hedgehog N Terminus ELISA Enzyme Linked Immunosorbent Assay kit is an in vitro enzyme linked immunosorbent assay for the quantitative measurement of mouse Shh N in serum plasma using EDTA and Citrate as an anticoagulant heparin is not recommended and cell culture supernatants This assay employs an antibody specific for mouse Shh N coated on a 96 well plate Standards and samples are pipetted into the wells and Shh N present in a sample is bound to the wells by the immobilized antibody The wells are washed and biotinylated anti mouse Shh N antibody is added After washing away unbound biotinylated antibody HRP conjugated streptavidin is pipetted to the wells The wells are ag2. 000 Mouse Shh N concentration pg ml Mouse Shh N concentration pg ml B SENSITIVITY The minimum detectable dose of Shh N is typically less than 5 pg ml C RECOVERY Recovery was determined by spiking various levels mouse Shh N into mouse serum plasma EDTA and citrate and cell culture media Mean recoveries are as follows Sample Type Average Recovery Range Serum 78 88 71 87 Plasma 77 03 71 79 Cell culture media 82 11 78 86 D LINEARITY Sample Type Serum Plasma Cell Culture Media 1 2 Average of Expected 99 00 90 69 84 65 Range 96 91 107 83 103 76 92 1 4 Average of Expected 92 99 85 01 79 86 Range 85 101 68 83 71 87 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 CD30 L CD30 T CD40 CRG 2 CTACK CXCL16 Eotaxin Eotaxin 2 Fas Ligand Fractalkine GCSF GM CFS IFN y IGFBP 3 IGFBP 5 IGFBP 6 IL 1 a IL 1f IL 2 IL 4 IL 9 IL 10 IL 13 KC Leptin R LEPTIN OB LIX L Selectin Lymphotactin MCP 1 MCP 5 M CSF MIG MIP 1a MIP 1y MIP 2 MIP 3f MIP 3a PF 4 P Selectin RANTES SCF SDF 1a TARC TCA 3 TECK TIMP 1 TNF RI TNF RIL TPO VCAM 1 VEGF X TROUBLESHOOTING GUIDE Problem Cause Solution 1 Poor standard curve 1 Inaccurate pipetting 2 Improper standard dilution Check pipettes 2
3. Ensure a brief spin of Item C and dissolve the powder thoroughly by a gentle mix 2 Low signal 1 Too brief incubation times 2 Inadequate reagent volumes or improper Ensure sufficient incubation time assay procedure step 2 may change to over night 2 Check pipettes and ensure correct dilution preparation 3 Large CV 1 Inaccurate pipetting 1 Check pipettes 4 High background 1 Plate is insufficiently 1 Review the manual washed for proper wash If using a plate washer check that all ports are unobstructed 2 Contaminated wash 2 Make fresh wash buffer buffer 5 Low sensitivity 1 Improper storage of the 1 Store your standard ELISA kit at lt 20 C after reconstitution others at 4 C Keep substrate solution protected from light 2 Stop solution 2 Stop solution should be added to each well before measure Note This product is for research use only ag 2 USA Abgent Inc Toll Free 888 735 7227 Or 858 875 1900 info_us abgent wuxiapptec com CHINA Abgent Suzhou 86 512 69369088 sales abgent wuxiapptec com EUROPE Abgent Europe 44 0 1235 854042 eurosales abgent wuxiapptec com For other countries www abgent com
4. ain washed a TMB substrate solution is added to the wells and color develops in proportion to the amount of Shh N bound The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm Il REAGENTS 1 Shh N Microplate Item A 96 wells 12 strips x 8 wells coated with anti mouse Shh N 2 Wash Buffer Concentrate 20x Item B 25 ml of 20x concentrated solution Standards Item C 2 vials of recombinant mouse Shh N 4 Assay Diluent A Item D 30 ml diluent buffer 0 09 sodium azide as preservative For Standard Sample serum plasma diluent 5 Assay Diluent B Item E 15 ml of 5x concentrated buffer For Standard Sample cell culture medium diluent 6 Detection Antibody Shh N Item F 2 vial of biotinylated anti mouse Shh N each vial is enough to assay half microplate 7 HRP Streptavidin Concentrate Item G 200 ul 300x concentrated HRP conjugated streptavidin 8 TMB One Step Substrate Reagent Item H 12 ml of 3 3 5 5 tetramethylbenzidine TMB in buffer solution Ww 9 Stop Solution Item I 8 ml of 0 2 M sulfuric acid Ill 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 stored for up to 1 month at 2 to 8 C Return unused wells to the pouch containing desicc
5. ant 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 Tubes to prepare standard or sample dilutions OAAYDNABWN 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 A Item D 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 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 Briefly spin the vial of Item C and then add 400 ul Assay Diluent A for serum plasma samples or 1x Assay Diluent B for cell culture supernates into Item C vial to prepare a 50 ng ml standard Dissolve the p
6. he vial Item G and pipette up and down to mix gently Add 40 ul of HRP Streptavidin concentrate into a tube with 12 ml 1x Assay Diluent B to prepare a 300 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 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 O
7. ne Step Substrate Reagent Item H to each well Incubate for 30 minutes at room temperature in the dark with gentle shaking Add 50 ul of Stop Solution Item I to each well Read at 450 nm immediately VII 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 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 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 E 3 E 1 c c z 2 II iT Q Q O 01 O 04 0 01 r r r 0 01 r r r 1 10 100 1 000 10 000 1 10 100 1 000 10
8. owder thoroughly by a gentle mix Add 20 ul Shh N standard 50 ng ml from the vial of Item C into a tube with 980 ul Assay Diluent A or 1x Assay Diluent B to prepare a 1 000 pg ml standard solution Pipette 300 ul Assay Diluent A or 1x Assay Diluent B into each tube Use the 1 000 pg ml 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 pg ml 20 ul standard 200 ul 1580 P 200u 2001 200l 200g M 200 ul BeBHHHHE 1 000 400 160 25 6 10 24 4 10 pg ml pg ml pg ml on pg ml pg ml pg ml E 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 pl of 1x 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 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 300 fold with 1x Assay Diluent B For example Briefly spin t
Download Pdf Manuals
Related Search