FTO (mouse intracellular) ELISA Kit
Contents
1. an Be sure that reagents were prepared correctly Omssioneuieadens and added in the correct order Hilntion rror Check pipetting technique and double check calculations FOR RESEARCH USE ONLY Not to be used on humans Fax 408 493 1801 tech biovision com Tel 408 493 1800 www biovision com Page 2 of 22. BioVision FTO mouse intracellular ELISA Kit Catalog K4922 100 100 assays Store kit at 4 C Description FTO Fat mass and obesity associated gene was discovered as a responsible gene causing the mouse fused toes mutation The predicted 502 amino acid Fto protein has a calculated molecular mass of 58 kD and contains an N terminal bipartite nuclear localization signal RT PCR detected Fto expression throughout mouse embryonic development and in all adult mouse tissues examined except heart and skin Bioinformatics analysis indicates that FTO shares sequence motifs with iron and 2 oxoglutarate 2OG dependent oxygenases Recombinant murine Fto catalyzes the iron and 20G dependent demethylation of 3 methylthymine in single stranded DNA with concomitant production of succinate formaldehyde and carbon dioxide In wildtype mice Fto mRNA is most abundant in the brain particularly in hypothalamic nuclei and are regulated by feeding and fasting This assay is a sandwich Enzyme Linked Immunosorbent Assay ELISA for quantitative determination of mouse FTO in cells A monoclonal antibody specific for FTO has been precoated onto the 96 well microtiter plate Standards and samples are pipetted into the wells for binding to the coated antibody After extensive washing to remove unbound compounds FTO is recognized by the addition of a purified polyclonal antibody specific for FTO Detection Antibody After removal of excess polyclonal antibo
3. a quadratic equation d Ifthe Test Samples were diluted multiply the interpolated values by the dilution factor to calculate the corrected mouse FTO concentrations 42 i y 0 7751x 2 5792x 0 0288 R2 4 2 oO gt 8 b Cone ng ml 0 1 2 3 OD at 450 nm Performance Characteristics Intra assay Precision 3 samples of known concentrations of mouse FTO were assayed in replicates 6 times to test precision within an assay sewer oe et we ng ml 79 109 2 901 3 667 6 56 022 1 767 3 154 6 13 506 0 302 2 236 6 Inter assay Precision 3 samples of known concentrations of mouse FTO were assayed in 7 separate assays to test precision between assays Mean CV ng ml 120 499 4 719 3 916 49 286 2 575 5 225 58 256 3 669 6 299 Recovery Samples were spiked with known concentrations of mouse FTO averaged 99 range from 95 to 105 Samples Average Range 96 865 95 105 recoveries 99 043 95 105 99 636 95 105 BioVision Incorporated 155 S Milpitas Boulevard Milpitas CA 95035 USA rev 07 15 For research use only Technical Hints and Limitations It is recommended that all standards QC sample and samples be run in duplicate Do not combine leftover reagents with those reserved for additional wells Reagents from the kit with a volume less than 100 ul should be centrifuged Residual wash liquid should be drained from the wells after last
4. dy HRP conjugated anti rabbit IgG Detector is added Following a final washing peroxidase activity is quantified using the substrate 3 3 5 5 tetramethylbenzidine TMB The intensity of the color reaction is measured at 450 nm after acidification and is directly proportional to the concentration of FTO in the samples This ELISA is specific for the measurement of natural and recombinant mouse FTO It does cross react with mouse adiponectin mouse RBP4 mouse Nampt mouse vaspin mouse progranulin mouse resistin mouse clusterin mouse GPX3 mouse CD137 rat adiponectin rat Nampt human adiponectin human Nampt human progranulin The assay range is 0 156 10 ng FTO ml and a detection limit of 20 pg ml based on adding two standard deviations to the mean value of the 50 zero standards Kit Contents Pre coated Microtiter Plate 1 ea 12 x 8 well strips K4922 100 1 Wash Buffer 10X 50 ml K4922 100 2 Diluent 5X 50 ml K4922 100 3 Lysis Buffer 10X 12 ml K4922 100 4 Detection Antibody 12 ml K4922 100 5 Detector 100X Hrp conjugated anti IgG 150 ul K4922 100 6 Mouse FTO Standard lyophilized 20 ng 1 vial K4922 100 7 Mouse FTO QC Sample lyophilized 1 vial K4922 100 8 TMB Substrate Solution 12 ml K4922 100 9 Stop Solution 12 ml K4922 100 10 Plate Sealers 3 each K4922 100 11 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 temp
5. eratures greater than 25 C Assay Procedure Read the ENTIRE Protocol Before Proceeding Test Samples Standards QC Sample We recommend these be run in duplicate a Cell Lysates Grow cells to 90 confluency scrape cells off the plate transfer to an appropriate tube Keep on ice and microcentrifuge at 1 200 rom for 5 min at 4 C Remove supernatant rinse cells once with ice cold PBS Remove PBS and add 200 ul ice cold 1X Lysis Buffer supplemented with 1 mM phenyl methylsulfonyl fluoride PMSF to ten million cells Incubate on ice for 30 min Microcentrifuge at 12 000 rom for 5 min at 4 C and transfer the supernatant to a new tube The supernatant is the cell lysate Use freshly prepared cell lysate samples Note Cell lysates have to be diluted in Diluent 1X Samples containing visible precipitates must be clarified before use As starting point 1 10 to 1 1000 dilutions are recommended BioVision Incorporated 155 S Milpitas Boulevard Milpitas CA 95035 USA d rev 07 15 For research use only QC Sample Reconstitute mouse FTO QC Sample with 1 ml of dH2zO0 Mix the QC Sample to ensure complete reconstitution Allow to sit for a minimum of 15 min The QC Sample is ready to use do not dilute it refer to the C of A for current QC Sample concentration Standards Reconstitute mouse FTO Standard with 1 ml of dH2O to produce a stock solution 20 ng ml Mix the Stock solution to ensure complete reconstitution Allow to sit for a minimu
6. m of 15 min The reconstituted standard should be aliquoted and stored at 20 C Prepare Standard Curve using 2 fold serial dilutions with 1X Diluent Toobtain Add S dnt 300 ul 300 ul 300 ul 300 ul 300 yl 300l 300 LI a YANAINANANANAN gt ka 20 10 5 2 5 1 25 0 625 0 3125 0 15625 0 ng ml ng ml ng ml ng ml ng ml ng ml ng ml ng ml ng ml 2 Reagent Preparation Prepare just the appropriate amounts for the assay a Tang 1X Wash Buffer Dilute 10X Wash Buffer 1 9 with dH2O to obtain 1X Wash Buffer 1X Diluent Dilute 5X Diluent 1 4 with dH2O to obtain 1X Diluent 1X Lysis Buffer Dilute 10X Lysis Buffer 1 9 with dH2O to obtain 1X Lysis Buffer 1X Detector Dilute 100X Detector 1 99 with 1X Diluent to obtain 1X Detector Detection Antibody amp TMB Substrate Solution Ready to use Warm to room temp before use Note The diluted Detector must be used within 1 hr of preparation 3 Assay Protocol a b Determine the number of 8 well strips needed for 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 Add 100 ul of the Standards Samples and QC Sample into the appropriate wells in duplicate Cover plate with plate sealer and incubate for 1 hr at 37 C Aspirate and wash x 3 with 300 ul of 1X Wash Buffer Add 100 ul Detection Antibody to each well and tap gently on the side of the plate to mix Cover plate
7. wash by tapping the plate on absorbent paper 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 Once reagents have been added to the 8 well strips DO NOT let the strips DRY at any time during the assay Keep Substrate Solution protected from light The Stop Solution consists of phosphoric acid Although diluted the Stop Solution should be handled with gloves eye protection and protective clothing Troubleshooting PROBLEM POSSIBLE CAUSES signal SOLUTIONS ae Check that all reagents have been added in the Washes too stringent Use an automated plate washer if possible No si k Incubation times Incubation times should be followed as o signal or Weak inadequate indicated in the manual late reader settings not Verify the wavelength and filter setting in the optimal plate reader Incorrect assay temperature Use recommended incubation temperature Bring substrates to room temperature before use Concentration of detector too high Use recommended dilution factor High background T Poor standard curve Unexpected results Ensure all wells are filling wash buffer and are Inadequate washing aspirated completely Wells not completely l aspirated Completely aspirate wells between steps Reagents poorly mixed Be sure that reagents are thoroughly mixed
8. with plate sealer and incubate for 1 hr at 37 C Aspirate and wash x 3 with 300 ul of 1X Wash Buffer Add 100 ul of the 1X Detector to each well Cover plate with plate sealer and incubate for 1 hr at 37 C Remove plate from 37 C aspirate and wash x 5 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 Add 100 ul of the TMB Substrate Solution to each well 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 Tel 408 493 1800 Fax 408 493 1801 www biovision com tech biovision com Page 1 of 2 BioVision Vi o Tap the plate gently to ensure thorough mixing The substrate reaction yields a blue solution that turns yellow when Stop Solution is added Caution Stop Solution is a Corrosive Solution p Measure the OD at 450 nm in an ELISA plate reader within 30 min Calculations a Average the duplicate readings for each Standard QC Sample and Test Sample and subtract the average blank value obtained with the 0 ng ml point b Generate a Standard Curve by plotting the average absorbance on the horizontal X axis vs the corresponding concentration ug ml on the vertical Y axis See Typical Data below c Calculate the Test Sample FTO concentrations by interpolation of the Standard Curve regression curve as shown below in the form of
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