NQO1 (human intracellular) ELISA Kit
Contents
1. BioVision NQO1 human intracellular ELISA Kit Catalog K4926 100 100 assays Store kit at 4 C Description NAD P H quinone acceptor oxidoreductase 1 NQO1 a multifunctional antioxidant enzyme and exceptionally versatile cytoprotector both protects the cell from carcinogenic oxidative damage and stabilizes the tumor suppressor p53 protein At high glycolysis levels NQO1 stabilizes and protects p53 from ubiquitin independent degradation a process that is NADH dependent and also elevates NAD NADH levels NAD and NADH play a crucial role in cellular energy metabolism and a dysregulated NAD NADH ratio is implicated in metabolic syndrome In humans NQO1 is expressed at high levels in adipocytes and its expression levels are positively correlated with adiposity glucose tolerance and liver dysfunction Thus NQO1 may provide the basis for a new therapy for the treatment of metabolic syndrome This assay is a sandwich Enzyme Linked Immunosorbent Assay ELISA for quantitative determination of human NQO1 in cells A monoclonal antibody specific for NQO1 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 NQO1 is recognized by the addition of a purified polyclonal antibody specific for NQO1 Detection Antibody After removal of excess polyclonal antibody HRP conjugated anti rabbit IgG Detector is adde2. al Hints and Limitations e tis 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 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 8 well strips DO NOT let the strips DRY at any time during the assay e Keep Substrate Solution protected from light e 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 SOLUTIONS ae Check that all reagents have been added in the Washes too stringent Use an automated plate washer if possible No sianal k Incubation times Incubation times should be followed as i or Weak inadequate indicated in the manual aigna 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 fa
3. ctor High background ia Inadequate washing Ensure all wells are filling wash buffer and are 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 Poor standard curve Unexpected results FOR RESEARCH USE ONLY Not to be used on humans Tel 408 493 1800 Fax 408 493 1801 www biovision com tech biovision com Page 2 of 2
4. d Following a final washing peroxidase activity is quantified using the substrate 3 3 5 5 tetramethyloenzidine TMB The intensity of the color reaction is measured at 450 nm after acidification and is directly proportional to the concentration of NQO1 in the samples This ELISA is specific for the measurement of natural and recombinant human NQO1 It does not cross react with human adiponectin human RBP4 human Nampt human vaspin human progranulin human resistin human clusterin human ANGPTL38 human CTRP5 human IL 33 human leptin human GPX3 human NMNAT2 human sirtuin 1 human FTO mouse Nampt rat Nampt The assay range is 0 313 20 ng NQO1 ml and a detection limit of 100 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 K4926 100 1 Wash Buffer 10X 50 ml K4926 100 2 Diluent 5X 50 ml K4926 100 3 Lysis Buffer 10X 12 ml K4926 100 4 Detection Antibody 12 ml K4926 100 5 Detector 100X Hrp conjugated anti IgG 150 ul K4926 100 6 Human NQO1 Standard lyophilized 40 ng 1 vial K4926 100 7 Human NQO1 QC Sample lyophilized 1 vial K4926 100 8 TMB Substrate Solution 12 ml K4926 100 9 Stop Solution 12 ml K4926 100 10 Plate Sealers 3 each K4926 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 temperatures grea
5. ion ng ml on the vertical Y axis See Typical Data below c Calculate the Test Sample NQO1 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 NQO1 concentrations rr z y atge G 57a 0 445 RI 0 aoe z0 a _ i ae E Pa Pa ral z 1a wt a m a E a maj j F o n5 1 1 6 2 GG e ASG rri Performance Characteristics Intra assay Precision 4 samples of known concentration of human NQO1 were assayed in replicates 8 times to test precision within an assay Samples Mean pg ml SD CV n A549 CELLS 9 156 0 442 4 828 a 8 o o HT 29 CELLS 2 465 0 158 6 424 a HepG2 CELLS 1 570 0 115 7 034 Oo 8 HeLa CELLS 4 242 0 108 2 555 FB Inter assay Precision 4 samples of known concentration of human NQO1 was assayed in 5 separate assays to test precision between assays Samples Mean yg ml SD CV n Recovery A human cell lysate was spiked with a known concentration of human NQO1 and the recovery averaged 96 range from 90 to 105 _Samples___ Average Range A549 CELLS 98 23 95 105 HT 29 CELLS 93 05 90 100 HepG2 CELLS 93 38 90 100 HeLa CELLS 99 47 95 105 BioVision Incorporated 155 S Milpitas Boulevard Milpitas CA 95035 USA rev 01 12 For research use only Technic
6. stituted standard should be aliquoted and stored at Prepare Standard Curve using 2 fold serial dilutions with 1X Diluent Toobtain Add Into 0 625 ng ml 300 ul of NQO1 1 25 ng ml 300 ul of 1X Diluent 0 3125 ng ml 300 ul of NQO1 0 625 ng ml 300 ul of 1X Diluent 300 ul of 1X Diluent Empty tube 300pl 300 ul 300yl 9300p 300 pl Ss 300 300 u l a VIN ANAINANINAIN sTo x 40 20 10 5 2 5 1 25 0 625 0 3125 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 b C d e 1X Wash Buffer Dilute 10X Wash Buffer 1 9 with dH20 to obtain 1X Wash Buffer 1X Diluent Dilute 5X Wash Buffer 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 Henao Antibody amp TMB Substrate Solution Ready to use Warm to room temp efore use Note The diluted Detector must be used within 1 hr of preparation 3 Assay Protocol a O Se oad 7 JO _ t xA Ar 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 4 100 ul of the Standards Samples and QC Sample into the appropriate wells in uplicate Cover plate with pla
7. te 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 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 20 min Stop the reaction by adding 100 ul of Stop Solution to each well 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 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 Vi 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 concentrat
8. ter 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 on ice 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 methylsulfony 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 b d rev 01 12 For research use only QC Sample Reconstitute human NQO1 QC Sample with 1 ml of dH20 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 human NQO1 Standard with 1 ml of dH2O to produce a stock solution 40 ng ml Mix the Stock solution to ensure complete reconstitution Allow to sit ae minimum of 15 min The recon
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