Manual - RayBiotech, Inc.
Contents
1. based on the competitive enzyme immunoassay principle In this assay a biotinylated NPY peptide is spiked into the samples and standards The samples and standards are then added to the plate where the biotinylated NPY peptide competes with endogenous unlabeled NPY for binding to the anti NPY antibody After a wash step any bound biotinylated NPY 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 NPY peptide and inversely proportional to the amount of endogenous NPY in the standard or samples A standard curve of known concentration of NPY peptide can be established and the concentration of NPY 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 D 4 e tk gt Capture antibody y 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 table2. below V Reagents Size Description epee Stay Stability NPY NPY microplate tem A Item A NPY microplate tem a wells 12 ee x 8 wells coated with Hronka month at aor a antibody Washi Burer eo 25 ml 25 mi of 20x concentrated soon 20X concentrated solution 1 month at month ata eo Item B ag NPY eae ene Item 2 vials of Te NPY Peptide 1 vial is ee not store and an ee to run each standard in duplicate ee Anti NPY ae vials of Lyopniized annev ppsa not store and ae Item N 2 vials of Lyophilized anti NPY avis or tyopnizea annev 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 OLR NPY Peptide 2 vials of oo Biotinylated NPY Peptide 1 eee not store and OLR F vial is oo to assay the whole plate eee 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 Req
3. 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 B This is your Working Stock of Item F Pipette up and down to mix gently The final concentration of biotinylated NPY will be 10 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 NPY will be 5 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 NPY will be 5 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 NPY will be 5 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 aentire vial of Item F to 10 ml Assay Diluent Second Dilution b Positive Control r R Perform a 2 fold dilution of c Sample 125 pl a Standards 2 ml 100 ul of Working P of Working Stock of Working Stock ilies a adele Item F aa of Stock Item F 100 Item F oe yl Final concentration ul Pr
4. or techsupport raybiotech com F Preparation of Wash 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 200 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 NPY 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 o
5. 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 NPY should still be 5 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 NPY is 5 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 NPY is 5 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 8X If you have any questions regarding the recommendended dilutions you may contact technical support at 888 494 8555
6. RayBio Human Mouse Rat Neuropeptide Y Enzyme Immunoassay Kit Catalog EIA NPY EIAM NPY EIAR NPY User Manual Last revised December 1 2015 Caution Extraordinarily useful information enclosed Ke RayBiotech 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 NPY 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 Neuropeptide Y NPY is a 36 amino acid peptide hormone found in the neural system and has an important role in obesity The main effect of Neuropeptide Y is increased food intake and decreased physical activity It also in
7. creases the proportion of energy stored as fat and blocks nociceptive signals to the brain In addition to its role in obesity Neuropeptide Y has been associated with a number of other physiologic processes in the brain including the regulation of energy balance memory and learning and epilepsy Animal studies strongly demonstrate that the stimulation of Neuropeptide Yergic activity via the administration of certain Neuropeptide Y agonists increases food intake compared to control animals The effects of Neuropeptide Yergic activity on food intake is also demonstrated by the blockade of certain Neuropeptide Y receptors Y1 and Y5 receptors which expectedly inhibited Neuropeptide Yergic activity thus decreases food intake For its role in obesity an increase in Neuropeptide Y is caused by high levels of glucocorticosteriods through directly activating type II glucocorticosteriods receptors and indirectly by abolishing the negative feedback of CRF on Neuropeptide Y synthesis and release Meanwhile obesity induced insulin resistance and the mutation of the leptin receptor ObRb results in the abolishment of other negative feedback mechanisms to regulate Neuropeptide Yergic activity and ultimately food intake High levels of Neuropeptide Y were also found in the cerebrospinal fluid of patients with anorexia nervosa ll General Description The RayBio NPY Enzyme Immunoassay EIA Kit is an in vitro quantitative assay for detecting NPY peptide
8. efly 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
9. epared Positive Assay Diluent Prepared Sample Control 5 ng ml C Preparation of Standards 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 NPY 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 NPY is 5 ng ml in all standards 8 Briefly centrifuge the vial of NPY Standard Item C Reconstitute with 10 ul of ddH 20 and briefly vortex if desired Pipette 8 ul of Item C and 792 ul of 5 ng ml biotinylated NPY 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 NPY standard 5 ng ml biotinylated NPY 9 To make the 100 ng ml standard pipette 50 ul of the 1000 ng ml NPY standard 10 into the tube labeled 100 ng ml Mix thoroughly Repeat this step with each successive concentration preparing a dilution series as shown in the illustration below Each time use 450 ul of biotinylated NPY and 50 ul of the prior concentration until the 100 pg ml is reached Mix each tube thoroughly before the next transfer soul 50l 50 ul 50 ul Ne 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
10. h 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 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 Bri
11. nly Cover wells and incubate for 2 5 hours at room temperature 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 NPY 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 densit
12. uired 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 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 NPY 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 NPY 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 NPY 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 NPY Item F 5 Briefly centrifuge the vial of Biotinylated NPY Item F and reconstitute with 20 ul
13. y 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 EIA NPY 1 B BO T T T T T 0 01 041 1 10 100 1000 10000 Peptide Concentration ng ml B Sensitivity The minimum detectable concentrations of NPY is 0 2 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 Key Blank Buffer Only Total Binding Biotin NPY 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 EIA kit is designed to detect active NPY 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 Lodis
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