ANGPTL6 (human) Serum ELISA Kit
Contents
1. range in plasma and serum from 50 to gt 800 ng ml from healthy donors 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 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 SOLUTIONS No signal or weak Incubation times signal Omission of key reagent Check that all reagents have been added in the correct order Washes too stringent Use an automated plate washer if possible Incubation times should be followed as inadequate indicated in the manual Plate reader settings not Verify the wavelength and filter setting in the optimal plate reader Incorrect assay Use recommended incubation temperature Bring substra2. to sit for a minimum of 15 min The reconstituted standard should be aliquoted and stored at 20 C Prepare 1X Diluent Dilute 5X Diluent 1 4 with dH2O Prepare Substrate Solution and Il Mix together in equal volumes within 15 minutes of use Prepare Standard Curve using 2 fold serial dilutions with 1X Diluent Toobtain Add S nto 300 pl of ANGPTL6 25 ng ml 300 yl of ANGPTL6 12 5 ng ml 300 yl of ANGPTL6 6 25 ng ml 300 ul of ANGPTL6 3 13 ng ml 300 pl of Diluent 1X 300 ul 300 ul 300 ul 300 ul 300 ul 300 ul 300 ul a5 E 200 100 50 25 12 5 6 25 3 13 1 556 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 1X Wash Buffer Dilute 10X Wash Buffer 1 9 with dH2O to obtain 1X Wash Buffer 1X Diluent Dilute 5X Wash Buffer 1 4 with dH2O to obtain 1X Diluent 1X Detector 100X HRP Labeled Streptavidin Dilute 100X Detector 1 99 with 1X Diluent to obtain 1X Detector Note The diluted Detector must be used within 1 hr of preparation 3 Assay Protocol a 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
3. 300 ul of 1X Wash Buffer Warm Detection Antibody to room temperature Add 100 ul 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 to each well of mixed substrate solution Allow the color to develop at room temperature in the dark for 30 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 www biovision com Fax 408 493 1801 tech biovision com Page 1 of 2 BioVision V 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 Ty
4. BioVision c ANGPTL6 human Serum ELISA Kit Catalog K4916 100 100 assays Store kit at 4 C Description Seven angiopoietin like proteins ANGPTLs share the characteristic protein structure of the angiopoietin family ANG but differ in their inability to bind antiopoietin receptor Tie 2 ANGPTL6 was originally named angiopoietin related growth factor AGF having an N terminal coiled coil like domain and a C terminal fibrinogen like domain both of which are conserved in ANG It is a circulating protein secreted by liver that induces angiogenesis by direct effect of epidermal ANGPTL6 on endothelial cells and proliferation of skin cells and thereby promotes wound healing 80 of Angptl6 mice died at about embryonic day 13 The surviving null mice developed marked obesity lipid accumulation in skeletal muscle and liver and insulin resistance accompanied by reduced energy expenditure relative to controls Conversely mice with constitutive overexpression of ANGPTL6 showed leanness and increased insulin sensitivity resulting from increased energy expenditure and were also protected from high fat diet induced obesity insulin resistance and non adipose tissue steatosis This assay is a sandwich Enzyme Linked lmmunosorbent Assay ELISA for quantitative determination of human ANGPTLE6 in biological fluids A monoclonal antibody specific for ANGPTL6 has been precoated onto the 96 well microtiter plate Standards and samples are pipet
5. Do not expose reagents to temperatures 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 Serum Use a serum separator tube Let samples clot at room temperature for 30 min before centrifugation for 20 min at 1000 x g Assay freshly prepared serum or store serum in aliquots at 20 C for future use Avoid repeated freeze thaw cycles b Plasma Collect using heparin EDTA or citrate as an anticoagulant Centrifugation for 15 min at 1000 x g within 30 min of collection Assay freshly prepared plasma or store in aliquots at 20 C for future use Avoid repeated freeze thaw cycles Note Serum Plasma Urine or Cell Culture Supernatant have to be diluted in Diluent 1X Samples containing visible precipitates must be clarified before use As starting point 1 10 dilution of serum or plasma are recommended QC Sample Reconstitute Human ANGPTL6 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 BioVision Incorporated 155 S Milpitas Boulevard Milpitas CA 95035 USA d rev 02 14 For research use only Standards Reconstitute human ANGPTL6 Standard with 1 ml of dH2O to produce a stock solution 200 ng ml Mix the Stock solution to ensure complete reconstitution Allow
6. pical Data below c Calculate the Test Sample ANGPTL6 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 ANGPTL6 concentrations Standard Optical Density hANGPTL6 ng ml mean Conc ng ml OD at 450 nm Performance Characteristics Intra assay precision Six samples of known concentrations of human ANGPTL6 were assayed in replicates 8 times to test precision within an assay Mean SD N n 178 45 588 330 8 662 79 1125 170 8 584 63 988 169 8 397 97 1292 325 8 739 63 1315 178 8 6 5635 87 15 8 Inter assay precision Six samples of known concentrations of human ANGPTL6 were assayed in 8 separate assays to test precision between assays Mean SD N n 17927 603 336 8 610 74 5220 855 8 24516 1044 42 8 74729 4871 652 8 397 97 1292 325 8 6 5128 4545 810 8 Recovery When samples serum are spiked with known concentrations of human ANGPTL6 the recovery averages 96 range from 80 to 105 Samples Average Recovery Range 88 77 85 95 101 17 95 105 100 43 95 105 BioVision Incorporated 155 S Milpitas Boulevard Milpitas CA 95035 USA rev 02 14 For research use only 4 Expected values ANGPTL6 levels
7. ted into the wells for binding to the coated antibody After extensive washing to remove unbound compounds ANGPTL6 is recognized by the addition of a purified polyclonal antibody specific for ANGPTL6 Detection Antibody After removal of excess 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 ANGPTL6 in the samples The assay range is 1 56 100 ng ml ANGPTL6 ml The lowest level of ANGPTL6 that can be detected by this assay is 1 2 ng ml Kit Contents Component 1 plate coated with human ANGPTL6 Antibody Wash Buffer 10X Diluent 5X Detection Antibody Detector 100X HRP Conjugated anti rabbit IgG 100 Assays Part Number 12 x 8 well strips K4916 100 1 50 ml K4916 100 2 50 ml K4916 100 3 12 ml K4916 100 4 1 vial K4916 100 5 200 ng K4916 100 6 1 vial K4916 100 7 6 ml K4916 100 8 6 ml K4916 100 9 12 ml K4916 100 10 3 sealers K4916 100 1 1 human ANGPTL6 Standard lyophilized human ANGPTL6 QC sample lyophilized Substrate Solution TMB Substrate Solution II Peroxidase Stop Solution 3 plate sealers plastic film Storage Conditions Reagents must be stored at 2 8 C when not in use Bring reagents to room temperature before use
8. tes to room temperature before u temperature se Concentration of E detector too high Use recommended dilution factor High background Poor standard curve Unexpected results Ensure all wells are filling wash buffer and are aspirated completely Wells not completely Completely aspirate wells between steps Reagents poorly mixed Be sure that reagents are thoroughly mixed ME Be sure that reagents were prepared correctly Omission of reagents and added in the correct order Check pipetting technique and double check calculations Inadequate washing Dilution error 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
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