Nampt (Visfatin/PBEF) (human) Intracellular ELISA Kit
Contents
1. reactivity in this assay 15 Recovery Human cell lysates were spiked with known concentrations of human Nampt The recovery averages were 98 range from 90 to 105 Samples __ _Average Recovery Range pS 96 36 95 105 102 62 95 105 BioVision Incorporated 155 S Milpitas Boulevard Milpitas CA 95035 USA rev 10 15 For research use only Technical Hints and Limitations e It is recommended that all standards and samples be run in duplicate e Do not combine leftover reagents with those reserved for additional wells e Reagents from the kit with a volume less than 100 ul should be centrifuged e Residual wash liquid should be drained from the wells after last wash by tapping the plate on absorbent paper e Crystals could appear in the 10X solution due to high salt concentration in the stock solutions Crystals are readily dissolved at room temperature or at 37 C before dilution of the buffer solutions e Once reagents have been added to the 16 well strips DO NOT let the strips DRY at any time during the assay e Keep TMB Substrate Solution protected from light e The Stop Solution consists of Sulfuric acid Although diluted the Stop Solution should be handled with gloves eye protection and protective clothing Troubleshooting PROBLEM POSSIBLE CAUSES SOLUTIONS Omission of kev reagent Check that all reagents have oe been added in the correct order Washes too stringent Use an automated plate2. BioVision Nampt Visfatin PBEF human Intracellular ELISA Kit Catalog K4909 100 100 assays Store kit at 4 C Description Nampt nicotinamide phosphoribosy ltransferase is the rate limiting enzyme of the mammalian NAD biosynthesis pathway The circulating NMN nicotinamide mononucleotide and NAD are taken up by beta cells and converted to NAD by Nmnat nicotinamide mononucleotide adenyltransferase and intracellular Nampt The Nampt plays a critical role in enhancing life span and protecting against oxidative damage The Nampt Visfatin PBEF human Intracelluar ELISA Kit is to be used for the in vitro quantitative determination of intracellular human Nampt Visfatin PBEF This assay is a sandwich ELISA which utilizies a 96 well microtiter plate which was pre coated with a monoclonal antibody and a purified polyclonal detection antibody A HRP conjugated anti IgG Detector and TMB 3 3 5 5 tetramethylbenzidine is added to generate a color intensity directly proportional to the concentration of Nampt in the samples This ELISA is specific for the measurement of natural and recombinant human Nampt It does not cross react with human adiponectin human resistin human RELM B human leptin human GPX3 human ANGPTL4 human FABP4 human ANGPTL6 human PAI1 The assay range is 0 25 16 ng Nampt ml and a detection limit of 30 pg ml based on adding two standard deviations to the mean value of the zero standard Kit Contents Pr
3. Solution is added Caution Stop Solution is Corrosive s Measure the OD at 450 nm in an ELISA plate reader within 30 min Tel 408 493 1800 Fax 408 493 1801 www biovision com tech biovision com Page 1 of 2 BioVision 3 Vi Calculations a Average the duplicate readings for each standard and Test Sample b Subtract the average 0 ng ml standard from each of the above c Generate a Standard Curve by plotting the average absorbances on the horizontal X axis vs the corresponding concentration ng ml on the vertical Y axis See Typical Data below d Calculate the Test Sample Nampt concentrations by interpolation of the Standard Curve regression curve as shown above in the form of a quadratic equation e Ifthe Test Samples were diluted multiply the interpolated values by the dilution factor to calculate the corrected human Nampt serum concentrations y 1 9549x 4 0353x 0 0724 R 0 999 18 16 14 12 Conc ng ml 1 1 5 2 2 9 OD at 450 nm Performance Characteristics Intra assay Precision 2 samples of known concentration were assayed in replicates 10 times to test precision within an assay i i 666 2733 10 24 21 Inter assay Precision 2 samples of known concentration were assayed in 6 separate assays to test precision between assays CV 402 6 740 6 Note Mouse Nampt shows weak cross reactivity in this assay 5 Rat Nampt shows weak cross
4. e coated Microtiter Plate 6x16 well strips K4909 100 1 Wash Buffer 10X 2x30 ml K4909 100 2 Diluent 10X 2x30 ml K4909 100 3 Detection Antibody 60 ul K4909 100 4 K4909 100 5 K4909 100 6 K4909 100 7 K4909 100 8 K4909 100 9 K4909 100 10 Detector 100X 150 ul Human Nampt Standard lyophilized 32 ng 1 vial TMB 12 ml Stop Solution 12 ml Plate Sealers 2 Lysis Buffer 10X 12 ml Storage Conditions Reagents must be stored at 2 8 C when not in use Bring reagents to room temperature before use Do not expose reagents to temperatures greater than 25 C Assay Procedure Read the ENTIRE protocol before proceeding Day 1 We recommend the Samples and Standards be run in duplicate a Cells Lysates Ice cold 1X Lysis Buffer and 1X Diluent are prepared by 1 9 dilutions with dH2O and placed on ice until needed Grow cells to 80 90 confluency Adherent cells can be scraped off plate and transferred to a tube suspension cells pipetted to appropriate tube Centrifuge at 700 1000 x g for 5 min at 4 C and carefully remove and discard supernatant Wash 1 2 times with ice cold PBS Add 200 ul ice cold 1X Lysis Buffer with 1 mM PMSF not inluded per 1 x 10 cells and allow to stand on ice for 30 min Centrifuge at 10K x g for 5 min at 4 C and transfer supernatant to a new tube The supernatant is the cell lysate and should be freshly prepared and diluted into 1X Diluent As a starting point 1 10 to 1 1000 dilutions are recommended If samp
5. in 1X Diluent 8 ul antibody 1992 ul 1X Diluent Diluted antibody cannot be stored c Remove plate from 4 C aspirate and wash 3 times with 300 ul of 1X Wash Buffer After last wash tap inverted plate on a stack of paper towels Complete removal of liquid is essential for good performance e Add 100 ul of Detection Antibody to each well f Cover plate with plate sealer and incubate for 1 hr at 37 C g After about 30 45 min prepare 1X Detector Dilute 100X Detector 1 99 with 1X Diluent 100 ul Detector 9 9 ml of 1X Diluent h Remove plate from 37 C aspirate and wash 3 times with 300 ul of 1X Wash Buffer i After last wash tap inverted plate on a stack of paper towels Complete removal of liquid is essential for good performance j Add 100 ul of 1X Detector to each well k Cover plate with plate sealer and incubate for 1 hr at 37 C I Warm the TMB Solution and Stop Solution to room temperature m Remove plate from 37 C aspirate and wash 5 times with 300 ul of 1X Wash Buffer n After last wash tap inverted plate on a stack of paper towels Complete removal of liquid is essential for good performance 0 Add 100 ul of TMB Solution to each well p Allow the color to develop at room temperature in the dark for 10 min Stop the reaction by adding 100 ul of Stop Solution to each well r Tap the plate gently to ensure thorough mixing The substrate reaction yields a blue product that turns yellow when Stop
6. les fall outside the assay range a lower or higher dilution may be required b Standards Reconstitute Human Nampt Standard with 1 ml of dH2O to produce a stock solution 32 ng ml Mix the Stock solution to ensure complete reconstitution Allow to sit for a minimum of 15 min The reconstituted standard should be aliquoted and stored at 20 C c Prepare Standard Curve using 2 fold serial dilutions with 1X Diluent BioVision Incorporated 155 S Milpitas Boulevard Milpitas CA 95035 USA rev 10 15 For research use only To obtain Add 16 ng ml 8 ng ml 2 ng ml 1 ng ml 300 ul of 1X Diluent 0 5 ng ml 300 ul of Nampt 1 ng ml 300 ul of 1X Diluent 300 ul of Nampt 0 5 ng ml INININANONAN gJ 32 16 8 4 2 1 0 5 0 25 0 ng ml ng ml ng ml ng ml ng ml ng ml ng ml ng ml ng ml 300 ul of 1X Diluent 300 ul of 1X Diluent 300 ul of 1X Diluent 300 ul of 1X Diluent 4 ng ml a Determine the number of 16 well strips needed for the assay and insert them into the frame for current use The extra strips should be resealed in the foil pouch and can be stored at 4 C for up to 1 month b Add 100 ul of the Standards and Samples into the appropriate wells in duplicate c Cover the plate with plate sealer and incubate at 4 C overnight Day 2 Note the Detector must be used within 1 hr of preparation a Prepare 1X Wash Buffer Dilute 10X Wash Buffer 1 9 with dH2O b Warm Detection Antibody to room temperature Dilute the antibody 1 250
7. washer if possible Incubation times should be followed as indicated in the manual No signal or weak signal Incubation times inadequate Plate reader settings not Verify the wavelength and filter optimal setting in the plate reader Use recommended incubation temperature Bring substrates to room temperature before use Concentration of detector too Use recommended dilution high factor Ensure all wells are filling wash buffer and are aspirated completely Wells not completely aspirated Completely aspirate wells between steps Reaaents poorly mixed Be sure that reagents are ae thoroughly mixed Be sure that reagents were prepared correctly and added in the correct order Dilution erct Check pipetting technique and double check calculations FOR RESEARCH USE ONLY Not to be used on humans Incorrect assay temperature High background Inadequate washing Poor standard curve Omission of reagents Unexpected results Tel 408 493 1800 Fax 408 493 1801 www biovision com tech biovision com Page 2 of 2
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