FTO (human intracellular) ELISA Kit
Contents
1. 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. at 2 8 C when not in use Bring reagents to room temperature d Aspirate and wash x 3 with 300 ul of 1X Wash Buffer before use Do not expose reagents to temperatures greater than 25 C e Add 100 ul Detection Antibody to each well and tap gently on the side of the plate to IV Assay Procedure Read the ENTIRE Protocol Before Proceeding mix l l 1 Test Samples Standards QC Sample We recommend these be run in duplicate Cover plate with plate sealer and incubate for 1 hr at 37 C h a Cell Lysates Grow cells to 90 confluency Scrap cells off the plate and transfer to an 9 Aan ae hie ete 300 pl of Oe aa Buffer appropriate tube Keep on ice and microcentrifuge at 1 200 rom for 5 min at 4 C y RN A cae gels oes ae pic eiamee Remove supernatant rinse cells once with ice cold PBS Remove PBS and add 200 ul j Rome dale from 37 C aspirate ies d aon x5 aah 300 ul of 1X Wash Buffer ice cold 1X Lysis Buffer supplemented with 1 mM phenyl methylsulfonyl fluoride k After last wash tap inverted plate on a stack of paper towels Complete removal of PMSF to ten million cells Incubate on ice for 30 min Microcentrifuge at 12 000 rom liquid is essential for good performance for 5 min at 4 C and transfer the supernatant to a new tube The supernatant is the cell Add 100 ul of the TMB Substrate Solution to each well lysate Use freshly prepared cell lysate samples Note Cell lysates have to be diluted m Allow the color to develop at roo
3. BioVision ane FTO human intracellular ELISA Kit b QC Sample Reconstitute human FTO QC Sample with 1 ml of dH20 Mix the QC 100 A o Sample to ensure complete reconstitution Allow to sit for a minimum of 15 min The QC n a a a a re Sample is ready to use do not dilute it refer to the C of A for current QC Sample concentration c Standards Reconstitute human 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 minimum of 15 min The reconstituted standard should be aliquoted and stored at I 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 FTO is widely expressed in a variety of human tissues 20 with highest levels in brain and pancreatic islets Bioinformatics analysis indicates inl ily iti that FTO shares sequence motifs with iron and 2 oxoglutarate 20G dependent ey ieee pia ee eee oxygenases The FTO human IntraCellular ELISA Kit is to be used for the in vitro Toobtain Add to __ quantitative determination of human FTO in cell lysates or cell based assays screening This assay is a sandwich Enzyme Linked Immunosorbent Assay ELISA for quantitative determination of human FTO in cel
4. amples of known concentrations of human FTO were assayed in replicates 6 times to test precision within an assay Smee ngm O ng ml THP 1 cells 31 976 1 789 5 585 Oo 6 Molt4 cells 262 277 6 146 2 343 FUG A549 cells 96 285 1 569 1 629 Se HepG2 cells 130 960 2 823 2 156 0o 6 Inter assay Precision 3 samples of known concentrations of human FTO were assayed in 4 separate assays to test precision between assays Mean CV ng ml Molt4 cells 226 599 20 899 9 223 A549 cells 92 102 8 076 8 768 HepG2 cells 99 771 4 978 4 989 Recovery Different human cell lysates were spiked with known concentrations of human FTO and the recoveries averaged 100 range from 95 to 105 Samples Average Range THP 1 cells 95 416 95 105 A549 cells 102 444 95 105 HepG2 cells 103 941 95 105 BioVision Incorporated 155 S Milpitas Boulevard Milpitas CA 95035 USA rev 01 12 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 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
5. 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 Use recommended incubation temperature Bring substrates to room temperature before use temperature 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 an Be sure that reagents were prepared correctly Omssioneuieadens and added in the correct order Hilntion rror Check pipetting technique and double check calculations
6. gent Preparation Prepare just the appropriate amounts for the assay r a 1X Wash Buffer Dilute 10X Wash Buffer 1 9 with dH2O to obtain 1X Wash Buffer Pre coated Microtiter Plate 1 ea 12 x 8 well strips K4921 100 1 b 1X Diluent Dilute 5X Wash Buffer 1 4 with dH2O to obtain 1X Diluent Wash Buffer 10X 50 ml K4921 100 2 c 1X Lysis Buffer Dilute 10X Lysis Buffer 1 9 with dH20 to obtain 1X Lysis Buffer Diluent 5X 50 ml K4921 100 3 d 1X Detector Dilute 100X Detector 1 99 with 1X Diluent to obtain 1X Detector Lysis Buffer 10X 12 ml K4921 100 4 e Detection Antibody amp TMB Substrate Solution Ready to use Warm to room temp Detection Antibody 12 ml K4921 100 5 before use Detector 100X Hrp conjugated anti IgG 150 ul K4921 100 6 Note The diluted Detector must be used within 1 hr of preparation Human FTO Standard lyophilized 20 ng 1 vial K4921 100 7 3 Assay Protocol Human FTO QC Sample lyophilized 1 vial K4921 100 8 a Determine the number of 8 well strips needed for assay and insert them into the frame TMB Substrate Solution 12 ml K4921 100 9 for current use The extra strips should be resealed in the foil pouch and can be stored Stop Solution 12 ml K4921 100 10 at 4 C for up to 1 month Plate Sealers 3 each K4921 100 11 b Add 100 ul of the Standards Samples and QC Sample into the appropriate wells in duplicate Ill Storage Conditions c Cover plate with plate sealer and incubate for 1 hr at 37 C Reagents must be stored
7. ls 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 Nam po 0 ng ml me Lan polyclonal antibody 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 300 ul 300 ul 300 ul 300 ul 300ul 6300ul 300 WI samples This ELISA is specific for the measurement of natural and recombinant human FTO It does cross react with human adiponectin human RBP4 human Z 4 Y AY Y 4 4 Nampt human vaspin human progranulin human resistin human clusterin human GPX3 human sirtuin 1 human IDO human IL 33 human ANGPTL3 human lt ANGPTL4 human FGF21 mouse progranulin mouse ANGPTL8 mouse leptin rat mi Nampt The assay range is 0 156 10 ng FTO ml and a detection limit of 50 pg ml a based on adding two standard deviations to the mean value of the 50 zero 20 standards 10 5 2 5 1 25 0 625 0 3125 0 15625 0 ll Kit Contents ng ml ng ml ng ml ng ml ng ml ng ml ng ml ng ml ng ml 2 Rea
8. m temperature in the dark for 10 min in Diluent 1X Samples containing visible precipitates must be clarified before use As n Stop the reaction by adding 100 ul of Stop Solution to each well starting point 1 10 to 1 1000 dilutions are recommended BioVision Incorporated Tel 408 493 1800 Fax 408 493 1801 155 S Milpitas Boulevard Milpitas CA 95035 USA 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 a quadratic equation d Ifthe Test Samples were diluted multiply the interpolated values by the dilution factor to calculate the corrected human FTO concentrations t MiiaT HAS c 114 mR 0 020 n gt OD m 450 Performance Characteristics Intra assay Precision 4 s
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