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1. 50 ul 8 SOnl 50 ul 50 ul 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 12 Refer to step 6b This is a 2 fold dilution of the Positive Control The final concentration of biotinylated Nesfatin should still be 10 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 Nesfatin is 10 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 the appropriate Assay Diluent 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 the appropriate Assay Diluent 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 Nesfatin is 10 ng ml Note Optimal sample dilution factors should be determined empirically however you may re
2. 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 step 7 to each well Incubate for 45 minutes at room temperature with gentle 10 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 Nesfatin 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
3. for binding to the anti Nesfatin antibody After a wash step any bound biotinylated Nesfatin 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 Nesfatin peptide and inversely proportional to the amount of endogenous Nesfatin in the standard or samples A standard curve of known concentration of Nesfatin peptide can be established and the concentration of Nesfatin 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 Storage Stay Stability Component Size Size Description After i Nesfatin Nesttin Microplate em A Item A estan Microplate tem a WOLS 1E SUIS TOWE Cogteg wl 1 month at month at aor eee antibody Wash Buffer ee ft monthatas ee Item B 25
4. RayBio Human Mouse Rat Nesfatin Enzyme Immunoassay Kit Catalog EIA NES EIAM NES EIAR NES 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 Nesfatin 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 Obesity which is characterized by excessive accumulation of adipose tissue in the body has become one of the greatest public health challenges Obesity is not only associated with health problems linked to increased weight de
5. 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 Assay Diluent Perform a 2 fold dilution of Working Stock Item F Final concentration 10 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 Nesfatin 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 Nesfatin is 10 ng ml in all standards 8 Briefly centrifuge the vial of Nesfatin Standard Item C Pipette 8 ul of Item C and 792 ul of 10 ng ml biotinylated Nesfatin 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 Nesfatin standard 10 ng ml biotinylated Nesfatin 9 To make the 100 ng ml standard pipette 50 ul of the 1000 ng ml Nesfatin 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 Nesfatin and 50 ul of the prior concentration until the 100 pg ml is reached Mix each tube thoroughly before the next transfer
6. croplate 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 Note Assay Diluent A should be used for dilution of samples Item F and Item C when testing plasma or serum samples 1X Assay Diluent B should be used for dilution of samples Item F and Item C when testing cell culture media or other sample types A Preparation of Plate and Anti Nesfatin 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 Nesfatin antibody vial Item N Then add 50 ul of 1X Assay Diluent B to the vial 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 Nesfatin antibody working solution which will be used in step 2 of Assay Procedure Secti
7. ding 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
8. ference below for recommended dilution factors for serum Human 4X Mouse 4X 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 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 Dilute the HRP Streptavidin concentrate 200 fold with 1X Assay Diluent B Note do not use Assay Diluent A for HRP Streptavidin preparation in step 17 VIII 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 Nesfatin 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
9. ml 25 mi of 20x concentrated soon 20X concentrated solution 1 month at 4 ft monthatas The first standard Standard Nesfatin Peptide 2 vials of Nesfatin Peptide 1 vial is enough to run 2 3 days at 4 C Item C each standard in duplicate Additional dilutions Do not store Anti Nesfatin a a Item N 2 vias ofantiNesiain of anti Nesfatin 1 month ata at 4 month ata 30 ml contains 0 09 sodium azide as Assay Diluent A Item D preservative Diluent for standards and serum or plasma 15 ml of 5X concentrated buffer Diluent for Assay Diluent B Item E standards cell culture media or other sample 1 month at 4 C types and HRP Streptavidin on enn Nesfatin Peptide 2 vials of a er Nesfatin Peptide 1 vial is 2 3 2s daysatae R 2s daysatae on enn F a er to assay the whole plate HRP ee 600 ee 200X concentrated HRP conjugated oe not store and Concentrate ee G ee oe Positive Control Item M Positive Control Item M Item M 1 1 vial of Positive Control of Positive 1 vial of Positive Control 2 3 at 2 3daysat4C TMB One Step Substrate 12 ml of 3 3 5 5 tetramethylbenzidine TMB i Reagent Item H buffer solution Stop Solution Item 1 8 8 ml of 0 2 M sulfuric acid of 0 2 M sulfuric acid daea Return unused wells to the pouch containing desiccant pack reseal along entire edge Vi NOOR WD 8 9 10 11 Vil Additional Materials Required Mi
10. on VIII 6 Note The following steps may be done during the antibody incubation procedure step 2 of Assay Procedure B Preparation of Biotinylated Nesfatin Item F 5 Briefly centrifuge the vial of Biotinylated Nesfatin Item F 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 the appropriate Assay Diluent This is your Working Stock of Item F Pipette up and down to mix gently The final concentration of biotinylated Nesfatin will be 20 ng ml a Second Dilution of Item F for Standards Add 2 ml of Working Stock Item F to 2 ml of the appropriate Assay Diluent The final concentration of biotinylated Nesfatin will be 10 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 Nesfatin will be 10 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 Nesfatin will be 10 ng ml 1 vialis enough to run the itemF whole plate First Dilution Add entire vial of Item F to Working Stock Item F 10 ml Assay Diluent Second Dilution 5 b Positive Control 100 ul of
11. pendent pressure overload on lung joints and bones but also a important risk factor for life threatening diseases such as cardiovascular diseases type 2 diabetes and certain cancers Nesfatin is a naturally occurring hormone produced by brain cells Japanese scientists discovered in 2006 that it is responsible for regulating appetite and producing body fat Excess Nesfatin in the brain leads to a loss of appetite less frequent hunger a sense of fullness and a drop in body fat and weight A lack of Nesfatin in the brain leads to an increase of appetite more frequent episodes of hunger an increase of body fat and weight and the inability to feel full This latter condition can be artificially induced by injecting an anti Nesfatin antibody into the brain The receptors of Nesfatin within the brain are not completely understood although they are thought to be contained in the hypothalamus and in the solitary nucleus where Nesfatin is believed to produced via peroxisome proliferator activated receptors PPARs ll General Description The RayBio Nesfatin Enzyme Immunoassay EIA Kit is an in vitro quantitative assay for detecting Nesfatin peptide based on the competitive enzyme immunoassay principle In this assay a biotinylated Nesfatin peptide is spiked into the samples and standards The samples and standards are then added to the plate where the biotinylated Nesfatin peptide competes with endogenous unlabeled Nesfatin
12. 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 Nesfatin EIA 120 100 o o B B0 0 4 e 20 T T T T T T 0 001 0 01 0 1 1 10 100 1000 10000 Peptide Concentration ng ml B Sensitivity The minimum detectable concentrations of Nesfatin is 147 pg ml or 3 09 pM 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 Nesfatin 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
13. with Item M XI Specificity This kit detects 420aa Nesfatin protein No other active isoforms have been reported Cross Reactivity This EIA kit shows no cross reactivity with any of the cytokines tested Ghrelin Angiotensin Il NPY and APC XIV Publications Citing This Product 1 The relation of serum nesfatin 1 level with metabolic and clinical parameters in obese and healthy children Species Human Sample Type Plasma 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 rea

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