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1. 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 cAMP Antibody 1 2 Equilibrate plate to room temperature before opening the sealed pouch Label removable 8 well strips as appropriate for your experiment Assay Diluent D Item K and 5X Assay Diluent B Item E should be diluted 5 fold with deionized or distilled water Briefly centrifuge the anti cAMP 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 cAMP 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 cAMP Item F 5 Briefly centrifuge the vial of Biotinylated cAMP 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 of the Item F vial into a tube containing 10 ml of 1X Assay Diluent D This is your Working Stock of Item F Pipette up and down to mix gently The final co
2. Anti cAMP wee vials ofLyophiized anticae D natstoreand not store and wee Item N 2 vials of Lyophilized anti cAMP avis ortyopitzedanicaup ont store and Assay Buen tem K Diluent Assay Buen tem K Item K 15 ml oe 5X concentrated buffer Diluent for 1 Imoma at month ato standards and oe 15 ml E 5X concentrated buffer Diluent for anti Assay Divent B tem E Diwan Assay Divent B tem E em cAMP antibody and E i month ata ad month ata eee cAMP Peptide 2 vials of Fe ee rt oer Biotinylated cAMP Peptide oe not store and eee F 1 vial is Fe ee rt oer to assay the whole plate oe HRP ee 600 So ee 200X concentrated HRP conjugated ee not store and ee Item G So ee ee Poste Cont emm Poste Cont emm Item M 2 vials of Lyophilized Positive vile of Lyophiized Posive Conch Se lle ee 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 nana 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 Absorbent paper Distilled or deionized water SigmaPlot software or other
3. J Med 2002 346 21 1623 30 8 Tschop M Smiley DL Heiman ML Ghrelin induces adiposity in rodents Nature 2002 407 6806 908 913 9 Kojima M Hosoda H Date Y Nakazato M Matsuo H Kangawa K Ghrelin is a growth hormone releasing acylated peptide from stomach Nature 1999 402 6762 656 60 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 pr
4. RayBio Human Mouse Rat Cyclic AMP Enzyme Immunoassay Kit Catalog EIA cAMP EIAM cAMP EIAR cAMP User Manual Last revised 5 Nov 15 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 cAMP 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 Cyclic adenosine monophosphate cAMP cyclic AMP or 3 5 cyclic adenosine monophosphate is a second messenger important in many biological processes cAMP is derived from adenosine triphosphate ATP and used for intracellular si
5. Standardi Standard1 Standard2__ Standard2 Standard3 Standard3 Standard4 Standard4 Standard5 SA23 SA23 XI Specificity This EIA kit is designed to only detect Cyclic AMP XIV Select EIA Publications 1 Plum L Lin HV Dutia R Tanaka J Aizawa KS et al The Obesity Susceptibility Gene Carboxypeptidase E Links FoxO1 Signaling in Hypothalamic Pro opiomelanocortin Neurons with Regulation of Food Intake Nature Med 2009 15 10 1195 1201 Ghrelin EIA EIA GHR 1 2 Hug C Lodish HF Visfatin a new adipokine Science 2005 307 5708 366 7 3 Kim MK Crystal structure of visfatin pre B cell colony enhancing factor 1 nicotinamide phosphoribosyltransferase free and in complex with the anti cancer agent FK 866 J Mol Biol 2006 362 1 66 77 4 Revollo J R et al The NAD biosynthesis pathway mediated by nicotinamide phosphoribosyltransferase regulates Sir2 activity in mammalian cells J Biol Chem 2004 279 50754 50763 5 Oh I S Shimizu H Satoh T et al Identification of nesfatin 1 as a satiety molecule in the hypothalamus Nature 2006 443 7112 709 12 6 Zhang J Ren P Avsian Kretchmer O Luo C Rauch R Klein C Hsueh A Obestatin a peptide encoded by the ghrelin gene opposes ghrelin s effects on food intake Science 2005 310 5750 996 9 7 Cummings D Weigle D Frayo R Breen P Ma M Dellinger E Purnell J Plasma ghrelin levels after diet induced weight loss or gastric bypass surgery N Engl
6. form 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 cAMP is 1 ng ml Note Optimal sample dilution factors should be determined empirically however you may contact technical support 888 494 8555 techsupport raybiotech com to obtain recommended dilution factors for serum F Preparation of Wash Buffer and HRP 14 15 16 17 If Item B 20X Wash Concentrate 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 Briefly centrifuge the HRP Streptavidin vial Item G before use Do not vortex this solution as it is sensitive to oxygen Dilute the HRP Streptavidin concentrate 200 fold with 1X Assay Diluent B Note do not use Assay Diluent D for HRP Streptavidin preparation in step 17 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 cAMP 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
7. gnal transduction in many different organisms such as transferring into cells the effects of hormones like glucagon and adrenaline which cannot pass through the plasma membrane cAMP functions through three main effectors PKA the guanine nucleotide exchange factor GEF EPAC and cyclic nucleotide gated ion channels The intracellular levels of cAMP are regulated by the balance between the activities of two enzymes adenylyl cyclase AC and cyclic nucleotide phosphodiesterase PDE Cyclic AMP has been shown to be involved in cell growth differentiation and general metabolism and it is important for many biological function especially in the cardiovascular nervous and immune systems The measurement of intracellular Cyclic AMP in tissues and cell cultures may help to provide a clearer understanding of the physiology and pathology of many disease states ll General Description The RayBio CAMP Enzyme Immunoassay EIA Kit is an in vitro quantitative assay for detecting cAMP peptide based on the competitive enzyme immunoassay principle In this assay a biotinylated cAMP peptide is spiked into the samples and standards The samples and standards are then added to the plate where the biotinylated cAMP peptide competes with endogenous unlabeled cAMP for binding to the anti cAMP antibody After a wash step any bound biotinylated cAMP then interacts with horseradish peroxidase HRP streptavidin which catalyzes a color development reactio
8. n The intensity of the colorimetric signal is directly proportional to the amount of captured biotinylated cAMP peptide and inversely proportional to the amount of endogenous CAMP in the standard or samples A standard curve of known concentration of cAMP peptide can be established and the concentration of cAMP peptide in the samples can be calculated accordingly ll How It Works Anti lgG antibody Bi ae Y Y pre coated on the plate Y Y Target molecule Biotinylated ry in sample Peptide w tk gt Capture antibody p pee is added to the wells Biotin peptide Standard w A Sample interact competitivly Ma a i for spots on the capture antibodies T Y on a i el 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 Storage Sabiny Stability cAMP cAMP Micropate tem A Item A cAMP micropiate tem a wells 12 ee x 8 wells coated with Geis tae month at aor eee antibody Wash Butter ee 25 ml 25 mi of 20x concentrated soluton 20X concentrated solution 1 month at ft monthata ee Item B Standard cAMP ee ene rae 2 vials of Te cAMP Peptide 1 vial is eee not store and Item ee ene rae Te to run each standard in duplicate eee
9. ncentration of biotinylated CAMP will be 2 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 D The final concentration of biotinylated CAMP will be 1 ng ml b Second Dilution of Item F for Positive Control Add 105 ul of Working Stock Item F to 105 ul of the prepared Positive Control Item M See section D for Positive Control preparation The final concentration of biotinylated cAMP will be 1 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 cAMP will be 1 ng ml Reconstitute Item F in 20 ul ddH 0 1 vial is enough to run Item F the whole plate Working Stock Item F b Positive Control First Dilution Add entire vial of Item F to 10 ml Assay Diluent Second Dilution Perform a 2 fold dilution of a Standards 2 ml c Sample 125 pl Control 105 ul of Working f F Working Stock iain F of Working Stock of Working Stock i E Item F 2 ml of Stock Item F 105 yl Item F 125 pl Final concentration Assay Diluent Prepared Positive Prepared Sample 1ing ml C Preparation of Standards 7 Label 6 microtubes with the following concentrations 1 000 pg ml 100 ng ml 10 ng ml 1 ng ml 0 1 ng ml and 0 ng ml Pipette 450 ul of biotinylated cAMP I
10. oduct is for research use only 2015 RayBiotech Inc
11. tem F working solution prepapred 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 cAMP is 1 ng ml in all standards 8 Briefly centrifuge the vial of cAMP Standard Item C Reconstitute with 10 ul of ddHo0 and briefly vortex if desired 50 ul 50 ul 50 ul 50 ul OS ISAS FS 1000 100 10 1 0 1 0 ng ml ng ml ng ml ng ml ng ml ng 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 dilution of the Positive Control The final concentration of biotinylated cAMP should still be 1 ng ml The Positive Control is a mouse serum sample 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 cAMP is 1 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 D 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 D b Per
12. 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 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 CAMP EIA B B0 0 01 0 1 1 10 100 1000 10000 cAMP Concentration ng ml B Sensitivity The minimum detectable concentrations of cAMP is 4 4 ng 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 SA16 SA16
13. 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 Discard the solution and wash 4 times as directed in Step 3 Add 100 ul of prepared HRP Streptavidin solution see Reagent Preparation 10 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 1 to each well Read at 450 nm immediately Assay Procedure Summary Prepare all reagents samples and standards as instructed Add 100 ul anti cAMP to each

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