Home

Manual

1. Item eee a to run each standard in duplicate ee Anti Visfatin ee vials ofLyophiized anVisiain pgo not store and ee Item N 2 vials of Lyophilized anti Visfatin avi or tyophiizea anv 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 AR Visfatin Peptide 2 vials of oe ae Biotinylated Visfatin ee not store and AR F e a 1 vial is enough to assay the whole plate ee HRP S 600 Se eee 100X concentrated HRP conjugated ee not store and S Item G Se 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 Absorbent paper Distilled or deionized water SigmaPlot software or other software which can perform four parameter logistic reg
2. 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 Visfatin 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 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 Visfatin is 10 ng ml Note Optimal sample dilution factors should be determined empirically however you may reference 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 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
3. 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 Visfatin 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 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 so
4. 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 Visfatin peptide and inversely proportional to the amount of endogenous Visfatin in the standard or samples A standard curve of known concentration of Visfatin peptide can be established and the concentration of Visfatin 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 Visfatin isan Microplate tm A Item A isin microplate tom a 2 eee ee 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 Visfatin eee 2 vials of a Visfatin Peptide 1 vial is ee not store and
5. 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 Visfatin 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 Visfatin 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 Visfatin will be 10 ng ml Reconstitute Item F in 20 ul ddH 0 1 vial is enough to run Item F the whole plate J First Dilution Working Stock Item F Add entire vial of Item F to 10 ml Assay Diluent Second Dilution a Standards 2 Perform a 2 fold dilution of Working Stock Item F Final concentration 10 ng ml 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 mlof Working Stock Item F 2 ml of Assay Diluent 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 Visfatin Item F working solution prepared
6. RayBio Human Mouse Rat Visfatin Enzyme Immunoassay Kit Catalog EIA VIS EIAM VIS EIAR VIS 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 Visfatin 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
7. 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 Visfatin is 10 ng ml in all standards 8 Briefly centrifuge the vial of Visfatin Standard Item C Reconstitute with 10 ul of ddH20O and briefly vortex if desired Pipette 8 ul of Item C and 792 ul of 10 ng ml biotinylated Visfatin 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 Visfatin standard 10 ng ml biotinylated Visfatin 9 To make the 100 ng ml standard pipette 50 ul of the 1000 ng ml Visfatin 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 Visfatin and 50 ul of the prior concentration until the 100 pg ml is reached Mix each tube thoroughly before the next transfer SsOnl 50l 50 ul 50 ul IS OS OS IS J55 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 dilution of the Positive Control The final concentration of biotinylated Visfatin should still be 10 ng ml The Positive Control is a cell culture media sample that serves as a system control
8. lution 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 Visfatin 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 regression models with standard concentration on the x axis and percentage of absorbance see calculation below on
9. nd survival in Solid Ehrlich Carcinoma bearing mice Pharmacological Reports 2014 Available online 8 August 2014 DOI 10 1016 j pharep 2014 07 010 Species Mouse Sample Type Tissue Lysate 3 Guzel S et al Visfatin Leptin and TNF Interrelated Adipokines in Insulin Resistant Clinical and Subclinical Hypothyroidism Endocrine Research Aug2013 Vol 38 Issue 3 p184 194 DOI 10 3109 07435800 2012 760588 Species Human Sample Type Serum 4 Mabrouk R et al Serum Visfatin Resistin and IL 18 in A Group of Egyptian Obese Diabetic and Non Diabetic Individuals THE EGYPTIAN JOURNAL OF IMMUNOLOGY Vol 20 1 2013 Species Human Sample Type Serum 5 Yanni D et al The role of leptin soluble leptin receptor adiponectin and visfatin in insulin sensitivity in preterm born children in prepubertal ages Cytokine 64 2013 448A A 453 http dx doi org 10 1016 j cyto 2013 04 034 Species Human Sample Type Serum 6 Gumus U et al Plasma visfatin levels in adolescents with polycystic ovary syndrome A prospective case control study Journal of Pediatric and Adolescent Gynecology Sept 2014 epub ahead of print DOI 10 1016 j jpag 2014 08 007 Species Human Sample Type Plasma For additional publications citing this product please contact technical support at 888 494 8555 or techsupport raybiotech com 14 XIII Troubleshooting Guide e Check pipettes Poor standard e Inaccurate pipetting Briefly centrifuge Item C and dissolve cu
10. 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 Visfatin an adipokine that is highly enriched in the visceral fat has increased expression level in plasma during the development of obesity implying its important role in obesity Visfatin also Known as PBEF and Nampt a 52 kDa cytoplasmic protein composed of 473 amino acids is expressed primarily in bone marrow muscle and liver tissue but can also be detected in placenta lung kidney and heart tissue In addition to its role in obesity visfatin also has other biological functions Visfatin is a nicotinamide phosphoribosyltransferase Nampt enzyme that catalyzes first step in the biosynthesis of NAD from nicotinamide Visfatin has also been reported to be a cytokine PBEF that promotes B cell maturation and inhibits neutrophil apoptosis ll General Description The RayBio Visfatin Enzyme Immunoassay EIA Kit is an in vitro quantitative assay for detecting Visfatin peptide based on the competitive enzyme immunoassay principle In this assay a biotinylated Visfatin peptide is spiked into the samples and standards The samples and standards are then added to the plate where the biotinylated Visfatin peptide competes with endogenous unlabeled Visfatin for binding to the anti Visfatin antibody After a wash step any bound biotinylated Visfatin
11. ression 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 Visfatin 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 Visfatin 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 Visfatin 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 Visfatin Item F 5 Briefly centrifuge the vial of Biotinylated Visfatin 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 B This is your Working Stock of Item F Pipette up and down to mix gently The final concentration of biotinylated Visfatin will be 20 ng ml a Second
12. rve 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
13. 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 VIS EIA 1 BA 001 01 1 10 100 1000 Vistatin Peptide Concentration nagimi B Sensitivity The minimum detectable concentrations of Visfatin is 0 778 ng ml or 6 82 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 Visfatin 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 Cross Reactivity This EIA kit shows no cross reactivity with any of the cytokines tested Ghrelin Nesfatin NPY and APC XIV Publications Citing This Product 1 Elsamanoudy A et al Endothelial Dysfunction and Insulin Resistance as Pathophysiologic Mechanisms in a Rat Model of Preeclampsia American Journal of Biochemistry and Biotechnology 6 3 172 180 2010 DOI 10 3844 ajbbsp 2010 172 180 Species Rat Sample Type Plasma 2 Abdin A et al Effect of propranolol on IL 10 visfatin Hsp70 iNOS TLR2 and survivin in amelioration of tumor progression a

Manual

Contents

Download Pdf Manuals

Related Search

Related Contents