Manual
Contents
1. 7 Label 6 microtubes with the following concentrations 1000 ng ml 100 ng ml 10ng ml 1 ng ml 100 pg ml and 0 pg ml Pipette 450 ul of biotinylated Apelin Item F working solution prepared in step 6a into each tube except the 1 000 ng ml leave this one empty It is very important to make sure the concentration of biotinylated Apelin is 50 ng ml in all standards 8 Briefly centrifuge the vial of Apelin Standard Item C Reconstitute with 10 ul of ddH20O and briefly vortex if desired Pipette 8 pl of Item C and 792 ul of 50 ng ml biotinylated Apelin working solution prepared in step 6a into the tube labeled 1000 ng ml Mix thoroughly This solution serves as the first standard 1 000 ng ml Apelin standard 50 ng ml biotinylated Apelin 9 To make the 100 ng ml standard pipette 50 ul of the 1000 ng ml Apelin standard into the tube labeled 100 ng ml Mix thoroughly 10 Repeat this step with each successive concentration preparing a dilution series as shown in the illustration below Each time use 450 ul of biotinylated Apelin and 50 ul of the prior concentration until the 100 pg ml is reached Mix each tube thoroughly before the next transfer 50 ul S50ul 50 ul 50 ul OS IS IS Is SBE 1000 100 10 1 100 0 ng ml ng ml ng ml ng ml pg ml pg ml D Positive Control Preparation 11 Briefly centrifuge the Positive Control vial Item M and reconstitute with 100 ul of ddH20 12 Refer to step 6b This is a 2 fold dilu2. or other software which can perform four parameter logistic regression models with standard concentration on the x axis and percentage of absorbance see calculation below on the y axis Draw the best fit curve through the standard points Percentage absorbance B blank OD Bo blank OD where B OD of sample or standard and Bo OD of zero standard total binding A Typical Data These standard curves are for demonstration only A standard curve must be run with each assay Apelin EIA B BO a T T T T T 0 01 0 1 1 10 100 1000 10000 Apelin Concentration ng ml B Sensitivity The minimum detectable concentrations of Apelin is 5 84 pg ml C Detection Range 0 1 1 000 ng ml D Reproducibility Intra Assay CV lt 10 Inter Assay CV lt 15 E Assay Diagram Recommended Plate Layout Key Blank Buffer Only Total Binding Biotin Apelin only Standard 1 1000 ng ml Standard 2 100 ng ml Standard 3 10 ng ml Standard 4 1 ng ml Standard 5 100 pg ml Pos Control Biotin with Item M XI Specificity This kit is designed to target the C terminus of the 77 aa apelin peptide and therefore is expected to detect all active forms of Apelin including Apelin 36 Apelin 31 Apelin 28 and Apelin 13 can be detected Cross Reactivity This kit shows no cross reactivity with any of the cytokines tested Ghrelin Nesfatin and NPY XIV Publications Citing This Product 1 Gileles Hillel A Alons
3. Adjustable 1 25 ml pipettes for reagent preparation 100 ml and 1 liter graduated cylinders Absorbent paper Distilled or deionized water SigmaPlot software or other software which can perform four parameter logistic regression models Tubes to prepare standard or sample dilutions Orbital shaker Aluminum foil Plastic wrap Reagent Preparation Keep kit reagents on ice during reagent preparation steps A Preparation of Plate and Anti Apelin Antibody 1 2 Equilibrate plate to room temperature before opening the sealed pouch Label removable 8 well strips as appropriate for your experiment 5X Assay Diluent B Item E should be diluted 5 fold with deionized or distilled water Briefly centrifuge the anti Apelin antibody vial Item N and reconsititute with 55 ul of 1X Assay Diluent B to prepare the antibody concentrate Pipette up and down to mix gently The antibody concentrate should then be diluted 100 fold with 1X Assay Diluent B This is your anti Apelin antibody working solution which will be used in step 2 of Assay Procedure Section VIII Note The following steps may be done during the antibody incubation procedure step 2 of Assay Procedure B Preparation of Biotinylated Apelin Item F 5 Briefly centrifuge the vial of Biotinylated Apelin Item F and reconstitute with 20 ul of ddH20 before use 6 See the image below for proper preparation of Item F Transfer the entire contents
4. 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 e Plate is insufficiently washed e Contaminated wash buffer Follow storage recomendations in e Improper storage of the sections IV and V Keep substrate ELISA kit solution protected from light e Stop solution 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
5. Buffer and HRP 14 If Item B 20X Wash Concentrate contains visible crystals warm to room temperature and mix gently until dissolved 15 Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to yield 400 ml of 1X Wash Buffer 16 Briefly centrifuge the HRP Streptavidin vial Item G before use 17 Dilute the HRP Streptavidin concentrate 100 fold with 1X Assay Diluent B Vill Assay Procedure 1 Keep kit reagents on ice during reagent preparation steps It is recommended that all standards and samples be run at least in duplicate 2 Add 100 ul of Anti Apelin Antibody Item N See Reagent Preparation step 3 to each well Incubate for 1 5 hours at room temperature with gentle shaking 1 2 cycle sec You may also incubate overnight at 4 C 3 Discard the solution and wash wells 4 times with 1X Wash Solution Buffer 200 300 ul each Washing may be done with a multichannel pipette or an automated plate washer Complete removal of liquid at each step is essential to good assay performance After the last wash remove any remaining Wash Buffer by aspirating or decanting Invert the plate and blot it against clean paper towels 4 Add 100 ul of each standard see Reagent Preparation Section C Positive Control see Reagent Preparation Section D and sample see Reagent Preparation Section E in appropriate wells Be sure to include a blank well Assay Diluent only Cover wells and incubate for 2 5 hours at room t
6. RayBio Human Mouse Rat Apelin C Terminus Enzyme Immunoassay Kit Catalog EIA APC EIAM APC EIAR APC User Manual Last revised December 1 2015 Caution Extraordinarily useful information enclosed Re RayBiotech 1 ii 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 Table of Contents amor SSSCS di i In oenemionewwin SSSCSCS d Cd m forne Cd ao paot ooo a ao Preparation A Preparation of Plate and Anti Apelin Antibody B Preparation of Biotinylated Peptide Item F C Preparation of Standards D Preparation of Positive Control E Preparation of Samples F Preparation of Wash Buffer and HRP Strep vm Assay Procedure lege fix Assay Procedure Summary O Assay Procedure Summary pe Calculation of Results A Typical Data B Sensitivity a See eee C Detection Range D Reproducibility E Assay Diagram x Specifcty OOOO O O Specificity Ce xu Select Publications OO O O Select Publications a Troubleshooting Guide Please read the entire manual carefully before starting your experiment I Introduction Apelin an endogenous ligand for the G protein coupled APJ receptor has been recently extensively studied in obesity research It is not only expressed in adipocyte tissue but also widely expressed in various other or
7. a month at aor a antibody Washi Burer ee 25 ml 25 mi of 20x concentrated soon 20X concentrated solution 1 month at month ata ee Item B Standard eS Peptide 2 vials of oT eee Apelin Peptide 1 vial is ee not store and Item eS oT eee to run each standard in duplicate ee Anti ie Polyclonal vials ofLyophiizedan Apein D nattoreand not store and ie Item N 2 vials of Lyophilized anti Apelin avis oFtyopitzed anapon ont store and 15 ml of 5X concentrated buffer Diluent for both 5X Assay Diluent B Item E standards and samples including serum plasma 1 month at 4 C cell culture media or other sample types o Apelin Peptide 2 vials of a uae Biotinylated Apelin Peptide e not store and o F 1 vial is a uae to assay the whole plate e HRP foe 600 eee 100X concentrated HRP conjugated ee not store and foe Item G eee ee Poste Convoi em M Poste Convoi em M Item M 1 vial of Lyophilized Positive 1 vial of Lyophilized Postive Conta e SOIS oe 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 TE mai ed Return unused wells to the pouch containing desiccant pack reseal along entire edge Vi NOOR OWOD 8 9 10 11 Vil Additional Materials Required Microplate reader capable of measuring absorbance at 450 nm Precision pipettes to deliver 2 ul to 1 ml volumes
8. e where the biotinylated Apelin peptide competes with endogenous unlabeled Apelin for binding to the anti Apelin antibody After a wash step any bound biotinylated Apelin then interacts with horseradish peroxidase HRP streptavidin which catalyzes a color development reaction The intensity of the colorimetric signal is directly proportional to the amount of captured biotinylated Apelin peptide and inversely proportional to the amount of endogenous Apelin in the standard or samples A standard curve of known concentration of Apelin peptide can be established and the concentration of Apelin peptide in the samples can be calculated accordingly ll How It Works Anti lgG antibody i ae Y Y pre coated on the plate Y Y Target molecule Biotinylated ry in sample Peptide t gt Capture antibody we is added to the wells Biotin peptide Standard w A Sample interact competitivly Ma a i for spots on the capture antibodies T Y BNP EIA Kit il Add HRP Streptavidin and Color Substrate Data Analysis IV Storage The entire kit may be stored at 20 C to 80 C for up to 6 months from the date of shipment For extended storage it is recommended to store at 80 C Avoid repeated freeze thaw cycles For prepared reagent storage see table below V Reagents Size Description epee Stay Stability Avein Microplate tem A Microplate Item A Avein microplate toma wells 12 ee x 8 wells coated with Hronk
9. emperature with gentle shaking 1 2 cycles sec overnight or at 4 C 5 Discard the solution and wash 4 times as directed in Step 3 Add 100 ul of prepared HRP Streptavidin solution see Reagent Preparation step 7 to each well Incubate for 45 minutes at room temperature with gentle shaking It is recommended that incubation time should not be shorter or longer than 45 minutes Discard the solution and wash 4 times as directed 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 1 2 cycles sec Add 50 ul of Stop Solution Item to each well Read at 450 nm immediately Assay Procedure Summary Prepare all reagents samples and standards as instructed Add 100 ul anti Apelin to each well Incubate 1 5 hours at room temperature or overnight at 4 C Add 100 ul standard or sample to each well Incubate 2 5 hours at room temperature or overnight at 4 C Add 100 ul prepared Streptavidin solution Incubate 45 minutes at room temperature Add 100 ul TMB One Step Substrate Reagent to each well Incubate 30 minutes at room temperature Add 50 ul Stop Solution to each well Read at 450 nm immediately X Calculation of Results Calculate the mean absorbance for each set of duplicate stands controls and samples and subtract the blank optical density Plot the standard curve using SigmaPlot software
10. gans such as the heart lung kidney gastrointestinal tract brain adrenal glands endothelium and human plasma Apelin is derived from a 77 amino acid prepropeptide that is cleaved into a 55 amino acid fragment and then into shorter forms The physiologically active form is thought to be apelin 36 although the pyroglutamylated form of apelin 13 which is also produced endogenously is more potent Studies have shown the association between apelin and obesity Apelin has higher circulating levels in obesity Insulin exerts a positive action on adipocyte apelin production Apelin also regulates fluid homeostasis playing an important role in the hypothalamic regulation of food and water intake and pituitary hormone release In addition to its role in obesity apelin acts as a mediator of cardiovascular control including for blood pressure and blood flow It is one of the most potent stimulators of cardiac contractility yet identified and plays a role in cardiac tissue remodeling Apelin levels are increased in left ventricles of patients with chronic heart failure and also in patients with chronic liver disease ll General Description The RayBio Apelin Enzyme Immunoassay EIA Kit is an in vitro quantitative assay for detecting Apelin peptide based on the competitive enzyme immunoassay principle In this assay a biotinylated Apelin peptide is spiked into the samples and standards The samples and standards are then added to the plat
11. o Alvares M Kheirandish Gozal L Peris E et al Inflammatory markers and obstructive sleep apnea in obese children The NANOS Study Mediators of Inflammation Accepted May 2014 http www hindawi com journals mi aip 605280 Epub ahead of print Species Human Sample Type Plasma 2 Than A Cheng Y Foh LC et al Apelin inhibits adipogenesis and lipolysis through distinct molecular pathways Mol Cell Endocrinol 2012 Oct 15 3862 1 2 227 41 doi 10 1016 j mce 2012 07 002 Species Mouse Sample Type Conditioned Media 3 Samy D Ismail C Deif A et al Induction of Apelin by Losartan in Renal Ischemia Reperfusion Injury in Rats Implication of Endothelial Nitric Oxide Synthase eNOS Phosphorylation J Phys Pharm Adv 2014 4 11 465 477 DOI 10 5455 jppa 20141 117043349 Species Rat Sample Type Tissue Lysate XIII Troubleshooting Guide e Check pipettes Poor standard e Inaccurate pipetting Briefly centrifuge Item C and dissolve curve e Improper standard dilution the powder thoroughly by gently mixing Briefly spin down vials before e Improper preparation of opening Dissolve the powder standard and or thoroughly biotinylated antibody Ensure sufficient incubation time e Too brief incubation times assay procedure step 2 may be done e Inadequate reagent overnight volumes or improper dilution Check pipettes and ensure correct preparation Low signal e Inaccurate pipetting Check pipettes e Air bubbles in wells e
12. of the Item F vial into a tube containing 10 ml of 1X Assay Diluent B This is your Working Stock of Item F Pipette up and down to mix gently The final concentration of biotinylated Apelin will be 100 ng ml a Second Dilution of Item F for Standards Add 2 ml of Working Stock Item F to 2 ml of 1X Assay Diluent B The final concentration of biotinylated Apelin will be 50 ng ml b Second Dilution of Item F for Positive Control Add 100 ul of Working Stock Item F to 100 ul of the prepared Positive Control Item M See section D for Positive Control preparation The final concentration of biotinylated Apelin will be 50 ng ml c Second Dilution of Item F for samples Add 125 ul of Working Stock Item F to 125 ul of prepared sample see section E for sample preparation This is a 2 fold dilution of your sample The final concentration of biotinylated Apelin will be 50 ng ml Reconstitute Item F in 20 ul ddH 0 1 vial is enough to run Item F the whole plate First Dilution Working Stock Item F Add entire vial of Item F to 10 ml Assay Diluent Second Dilution b Positive Control 100 ul of Working Stock Item F 100 ul Prepared Positive Control c Sample 125 ul of Working Stock Item F 125 ul Prepared Sample a Standards 2 ml of Working Stock Item F 2 ml of Final concentration Assay Diluent 50 ng ml Perform a 2 fold dilution of Working Stock Item F C Preparation of Standards
13. tion of the Positive Control The final concentration of biotinylated Apelin should still be 50 ng ml The Positive Control is a cell culture media sample that serves as a system control to verify that the kit components are working The resulting OD will not be used in any calculations if no positive competition is observed please contact RayBiotech Technical Support The Positive Control may be diluted further if desired but be sure the final concentration of biotinylated Apelin is 50 ng ml E Sample Preparation 13 If you wish to perform a 2 fold dilution of your sample proceed to step 6c If you wish to perform a higher dilution of your sample dilute your sample with 1X Assay Diluent B before performing step 6c EXAMPLE to make a 4 fold dilution of sample a Dilute sample 2 fold 62 5 ul of sample 62 5 ul of 1X Assay Diluent B b Perform step 6c 125 ul of working solution Item F 125 ul of sample prepared above The total volume is 250 ul enough for duplicate wells on the microplate It is very important to make sure the final concentration of the biotinylated Apelin is 50 ng ml Note Optimal sample dilution factors should be determined empirically however you may reference below for recommended dilution factors for serum Human 2X Mouse 2X Rat 2X If you have any questions regarding the recommendended dilutions you may contact technical support at 888 494 8555 or techsupport raybiotech com F Preparation of Wash
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