Manual - RayBiotech, Inc.
Contents
1. 10 pg ml 1 pg ml 0 1 pg ml and 0 pg ml Pipette 450 ul of biotinylated Glucagon Item F working solution prepapred in step 6a into each tube except the 1 000 pg ml leave this one empty It is very important to make sure the concentration of biotinylated Glucagon is 40 pg ml in all standards 8 Briefly centrifuge the vial of Glucagon Standard Item C Reconstitute with 10 ul of ddH20 and briefly vortex if desired Pipette 8 ul of Item C and 792 ul of 40 pg ml biotinylated Glucagon working solution prepared in step 6a into the tube labeled 1000 pg ml Mix thoroughly This solution serves as the first standard 1000 pg ml Glucagon standard 40 pg ml biotinylated Glucagon 9 To make the 100 pg ml standard pipette 50 ul of the 1000 pg ml Glucagon standard into the tube labeled 100 pg 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 Glucagon and 50 ul of the prior concentration until the 0 1 pg ml is reached Mix each tube thoroughly before the next transfer soul SOul 50 ul 50 ul I OS IS Os SEBEHE 1000 100 pg ml pg ml a i oe ee 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 dilution of the Positive Control The final concentration of biotinylated Glucagon should still be2. 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 Glucagon 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 or other software which can perform four parameter logistic regr
3. 130 E143DOI 10 1152 ajpendo 00344 2014 Species Mouse Sample Type Conditioned Media 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 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
4. 40 pg 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 Glucagon is 40 pg 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 Glucagon is 40 pg 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 Buffer and HRP 14 15 16 17 If Item B 20X Wash Concentrate contains vi
5. RayBio Human Mouse Rat Glucagon Enzyme Immunoassay Kit Catalog EIA GLU EIAM GLU EIAR GLU 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 ease SSSCS di i In oenemionsowin SSSCSCS d Cd m forne Cd ao paot ooo a ao Preparation A Preparation of Plate and Anti Glucagon 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 Glucagon is a 29 amino acid peptide hormone secreted by the pancreas Its function is to raise blood glucose levels to opposing the effect of insulin which lowers blood glucose levels Glucagon is synthesized and secreted f
6. ars eerie ual Peptide 1 vial is ee not store and Item eee Par ears eerie ual to run each standard in duplicate ee Anti ae Polyclonal vials ofLyophiizedant Gicagon D2 Soreand not store and ae Item N 2 vials of Lyophilized anti Glucagon avi or Lyophiizeaant atucagon 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 Biotinylated ae 2 vials of Lyophilized Biotinylated eG eer er ee ee ee not store and ae Item F eG eer er ee ee 1 vial is enough to assay the whole plate ee HRP oe 600 eee 200X concentrated HRP conjugated ee not store and oe 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 Adjustable 1 25 ml pipettes for reagent preparation 100 ml and 1 liter graduated cylinders Absorbe
7. ession 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 GLU EIA 1 120 ki N SA 4 B BO YO T T T T T 0 01 0 1 1 10 100 1000 10000 Glucagon Peptide Concentration pg ml B Sensitivity The minimum detectable concentrations of Glucagon is 4 77 pg ml C Detection Range 1 1 000 pg 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 Glucagon only Standard 1 1000 pg ml Standard 2 100 pg ml Standard 3 10 pg ml Standard 4 1 pg ml Standard 5 0 1 pg ml Pos Control Biotin with Item M XI Specificity Cross Reactivity This EIA kit shows no cross reactivity with any of the cytokines tested Ghrelin Nesfatin Angiotensin Il NPY and APC XIV Publications Citing This Product 1 Elliot A Ustione A Piston D Somatostatin and insulin mediate glucose inhibited glucagon secretion in the pancreatic cell by lowering cAMP American Journal of Physiology Endocrinology and Metabolism 15 January 2015 Vol 308 no 2 E
8. k of Item F Pipette up and down to mix gently The final concentration of biotinylated Glucagon will be 80 pg 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 Glucagon will be 40 pg 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 Glucagon will be 40 pg 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 Glucagon will be 40 pg 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 Perform a 2 fold dilution of Working Stock Item F c Sample 125 ul of Working Stock Item F 125 ul Prepared Sample b Positive Control 100 ul of Working Stock Item F 100 ul Prepared Positive Control a Standards 2 ml of Working Stock Item F 2 ml of Assay Diluent Final concentration 40 pg ml C Preparation of Standards 7 Label 6 microtubes with the following concentrations 1000 pg ml 100 pg ml
9. nt 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 Glucagon 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 Glucagon 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 Glucagon 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 Glucagon Item F 5 Briefly centrifuge the vial of Biotinylated Glucagon Item F and reconstitute with 20 ul of ddH2O before use 6 See the image below for proper preparation of Item F Transfer the entire contents of the Item F vial into a tube containing 10 ml of 1X Assay Diluent B This is your Working Stoc
10. rom alpha cells of the endocrine portion of the pancreas In rodents the alpha cells are located in the outer rim of the islet However alpha cells in human pancreas are distributed throughout the islet Glucagon and insulin are part of a feedback system that keeps blood glucose levels at a stable level The pancreas releases glucagon when glucose levels fall too low Glucagon causes the liver to convert stored glycogen into glucose which is released into the bloodstream Glucagon raises blood glucose levels High blood glucose levels stimulate the release of insulin Insulin allows glucose to be taken up and used by insulin dependent tissues Glucagon has important clinical applications Abnormally elevated levels of glucagon may be caused by pancreatic tumors such as glucagonoma with symptoms including necrolytic migratory erythema reduced amino acids and hyperglycemia It may occur alone or in the context of multiple endocrine neoplasia type 1 MENI ll General Description The RayBio Glucagon Enzyme Immunoassay EIA Kit is an in vitro quantitative assay for detecting Glucagon peptide based on the competitive enzyme immunoassay principle In this assay a biotinylated Glucagon peptide is spiked into the samples and standards The samples and standards are then added to the plate where the biotinylated Glucagon peptide competes with endogenous unlabeled Glucagon for binding to the anti Glucagon antibody After a wash step any bo
11. sible 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 Briefly centrifuge the HRP Streptavidin vial Item G before use Dilute the HRP Streptavidin concentrate 200 fold with 1X Assay Diluent B Vill Assay Procedure Keep kit reagents on ice during reagent preparation steps It is recommended that all standards and samples be run at least in duplicate Add 100 ul of Anti Glucagon 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 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 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 temperature with gentle shaking 1 2 cycles sec overnight or at 4 C
12. und biotinylated Glucagon 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 Glucagon peptide and inversely proportional to the amount of endogenous Glucagon in the standard or samples A standard curve of known concentration of Glucagon peptide can be established and the concentration of Glucagon peptide in the samples can be calculated accordingly Ill How It Works Y Y ace yi yi Target molecule Biotinylated tt in sample Peptide bed tk gt Capture antibody yi yi pn is added to the wells Biotin peptide Standard w 5 Sample interact competitivly m_a i for spots on the capture antibodies Y Y bii a 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 Glucagon Micropate ttem A Microplate Item A Giucagon crop tem a WEIS UA e Sele code win 1 month at month at aor a antibody Wash Butter ee 25 ml 25 mi of 20x concentrated soon 20X concentrated solution 1 month at month ata ee Item B Standard eee Peptide 2 vials of Lyophilized Par e
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