Mouse EGF ELISA Kit
Contents
1. nm immediately If wavelength correction is available subtract readings at 570 nm from those at 450 nm to correct optical imperfections Otherwise read the plate at 450 nm only Please note that some unstable black particles may be generated at high concentration points after stopping the reaction for about 10 minutes which will reduce the readings Data Analysis e Calculate the mean value of the duplicate or triplicate readings for each standard and sample e To generate a standard curve plot the graph using the standard concentrations on the x axis and the corresponding mean 450 nm absorbance on the y axis The best fit line can be determined by regression analysis using four parameter or log log logistic curve fit e Determine the unknown sample concentration from the Standard Curve and multiply the value by the dilution factor Standard Curve e The curve is provided for illustration only A standard curve should be generated each time the assay is performed Mouse EGF Standard Curve 1 04 OD 450 nm 0 1 1 n 1 107 107 10 mEGF ng ml Sensitivity and Specificity e The minimum detectable dose of mouse EGF is typically 0 03 ng ml e Intra assay and inter assay coefficients of variation were 4 9 and 7 1 respectively Linearity Average Percentage of Expected Value Sample Dilution Urine 1 250 88 1 500 97 1 1000 103 Recovery Standard Added Value 0 06 1 0 ng ml Recove2. 4 assaPro AssayMax Mouse EGF ELISA Kit Assaypro LLC 3400 Harry S Truman Blvd St Charles MO 63301 T 636 447 9175 F 636 395 7419 WWW assaypro com For any questions regarding troubleshooting or performing the assay please contact our support team at support assaypro com Thank you for choosing Assaypro Assay Summary Step 1 Add 50 ul of Standard or Sample per well Incubate 2 hours Step 2 Wash then add 50 ul of Biotinylated Antibody per well Incubate 2 hours Step 3 Wash then add 50 ul of SP Conjugate per well Incubate 30 minutes Step 4 Wash then add 50 ul of Chromogen Substrate per well Incubate 12 minutes Step 5 Add 50 ul of Stop Solution per well Read at 450 nm immediately Symbol Key BE Consult instructions for use Assay Template 12 11 10 Mouse Epidermal Growth Factor EGF ELISA Kit Catalog No EME2011 1 Sample insert for reference use only Introduction Human epidermal growth factor EGF is a mitogenic growth factor that plays important roles in cell growth proliferation and differentiation EGF is synthesized as a large precursor 1207 amino acids 134 kDa that is cleaved into a small mature protein 53 amino acids 6 kDa The precursor has 66 identity with the corresponding mouse protein 1 3 EGF binds to the cell surface receptor EGFR leading to the phosphorylation of the recep
3. ed at room temperature 20 25 C e Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccants inside Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator e Add 50 ul of Mouse EGF Standard or sample per well Cover wells with a sealing tape and incubate for 2 hours Start the timer after the last addition e Wash five times with 200 ul of Wash Buffer manually Invert the plate each time and decant the contents hit 4 5 times on absorbent material to completely remove the liquid If using a machine wash six times with 300 ul of Wash Buffer and then invert the plate decanting the contents hit 4 5 times on absorbent material to completely remove the liquid e Add 50 ul of Biotinylated Mouse EGF Antibody to each well and incubate for 2 hours e Wash the microplate as described above e Add 50 ul of Streptavidin Peroxidase Conjugate to each well and incubate for 30 minutes Turn on the microplate reader and set up the program in advance e Wash the microplate as described above e Add 50 ul of Chromogen Substrate per well and incubate for 12 minutes or till the optimal blue color density develops Gently tap plate to ensure thorough mixing and break the bubbles in the well with pipette tip e Add 50 ul of Stop Solution to each well The color will change from blue to yellow e Read the absorbance on a microplate reader at a wavelength of 450
4. iluent 1x may be stored for up to 30 days at 2 8 C e Store Standard at 2 8 C before reconstituting with Diluent and at 20 C after reconstituting with Diluent Other Supplies Required e Microplate reader capable of measuring absorbance at 450 nm Pipettes 1 20 ul 20 200 ul 200 1000 ul and multiple channel e Deionized or distilled reagent grade water Sample Collection Preparation and Storage e Cell Culture Supernatants Centrifuge cell culture media at 3000 x g for 10 minutes to remove debris Collect supernatants and assay Store the remaining samples at 20 C or below Avoid repeated freeze thaw cycles e Urine Collect urine using sample pot Centrifuge samples at 800 x g for 10 minutes Dilute samples 1 500 into EIA Diluent and assay If necessary dilute samples within the range of 1 250 to 1 1000 The undiluted samples can be stored at 20 C or below for up to 3 months Avoid repeated freeze thaw cycles Reagent Preparation e Freshly dilute all reagents and bring all reagents to room temperature before use e EIA Diluent Concentrate 10x If crystals have formed in the concentrate mix gently until the crystals have completely dissolved Dilute the EIA Diluent Concentrate 1 10 with reagent grade water Store for up to 30 days at 2 8 C e Standard Curve Reconstitute the 4 ng of Mouse EGF Standard with 2 ml of EIA Diluent to generate a 2 ng ml standard solution Allow the standard to sit for 10 minutes with gen
5. ould not be used beyond the expiration date Reagents e Mouse EGF Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against mouse EGF e Sealing Tapes Each kit contains 3 precut pressure sensitive sealing tapes that can be cut to fit the format of the individual assay e Mouse EGF Standard Mouse EGF in a buffered protein base 4 ng lyophilized e Biotinylated Mouse EGF Antibody 50x A 50 fold concentrated biotinylated polyclonal antibody against EGF 140 pul e EIA Diluent Concentrate 10x A 10 fold concentrated buffered protein base 30 ml e Wash Buffer Concentrate 20x A 20 fold concentrated buffered surfactant 30 ml 2 bottles e Streptavidin Peroxidase Conjugate SP Conjugate A 100 fold concentrate 80 ul e Chromogen Substrate A ready to use stabilized peroxidase chromogen substrate tetramethylbenzidine 8 ml e Stop Solution A 0 5 N hydrochloric acid to stop the chromogen substrate reaction 12 ml Storage Condition e Upon arrival immediately store components of the kit at recommended temperatures up to the expiration date e Store SP Conjugate and Biotinylated Antibody at 20 C e Store Microplate Diluent Concentrate 10x Wash Buffer Stop Solution and Chromogen Substrate at 2 8 C e Unused microplate wells may be returned to the foil pouch with the desiccant packs and resealed May be stored for up to 30 daysina vacuum desiccator e D
6. ry 84 101 Average Recovery 97 Cross Reactivity Species Cross Reactivity Canine 10 Bovine None Monkey 5 Mouse 100 Rat None Rabbit None Human None References 1 Carpenter G 1981 Handbook of Experimental Pharmacology 57 89 132 2 Taylor JM et al 1972 J Biol Chem 247 5928 5934 3 Bell Gl et al 1986 Nucleic Acids Res 14 21 8427 8446 4 Heist RS and Christiani D 2009 Pharmacogenomics 10 1 59 68 5 Markman B et al 2010 Adv Clin Chem 51 71 119 6 Renouf D and Moore M 2010 Expert Rev Anticancer Ther 10 4 529 540 Version 2 1R3 www assaypro com e e mail Support assaypro com
7. tle agitation prior to making dilutions Prepare duplicate or triplicate standard points by serially diluting the standard solution 2 ng ml 1 2 with EIA Diluent to produce 1 0 5 0 25 0 125 0 0625 and 0 0313 ng ml solutions EIA Diluent serves as the zero standard 0 ng ml Any remaining solution should be frozen at 20 C and used within 30 days Standard Point Dilution Mouse EGF ng ml P1 Standard 2 ng ml 2 0000 P2 1 part P1 1 part EIA Diluent 1 0000 P3 1 part P2 1 part EIA Diluent 0 5000 P4 1 part P3 1 part EIA Diluent 0 2500 P5 1 part P4 1 part EIA Diluent 0 1250 P6 1 part P5 1 part EIA Diluent 0 0625 P7 1 part P6 1 part EIA Diluent 0 0313 P8 EIA Diluent 0 0000 e Biotinylated Mouse EGF Antibody 50x Spin down the antibody briefly and dilute the desired amount of the antibody 1 50 with EIA Diluent Any remaining solution should be frozen at 20 C e Wash Buffer Concentrate 20x If crystals have formed in the concentrate mix gently until the crystals have completely dissolved Dilute the Wash Buffer Concentrate 1 20 with reagent grade water e SP Conjugate 100x Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 1 100 with EIA Diluent Any remaining solution should be frozen at 20 C Assay Procedure e Prepare all reagents standard solutions and samples as instructed Bring all reagents to room temperature before use The assay is perform
8. tor tyrosine kinase and subsequent activation of the signal transduction pathway 4 6 Principle of the Assay The AssayMax Mouse Epidermal Growth Factor ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of mouse EGF in urine and cell culture samples This assay employs a quantitative sandwich enzyme immunoassay technique that measures mouse EGF in less than 5 hours A polyclonal antibody specific for mouse EGF has been pre coated onto a 96 well microplate with removable strips EGF in standards and samples is sandwiched by the immobilized antibody and the biotinylated polyclonal antibody specific for mouse EGF which is recognized by a streptavidin peroxidase conjugate All unbound material is washed away and a peroxidase enzyme substrate is added The color development is stopped and the intensity of the color is measured Caution and Warning e This product is for Research Use Only and is Not For Use In Diagnostic Procedures Prepare all reagents working diluent buffer wash buffer standard biotinylated antibody and SP conjugate as instructed prior to running the assay e Prepare all samples prior to running the assay The dilution factors for the samples are suggested in this insert However the user should determine the optimal dilution factor e Spin down the SP conjugate vial and the biotinylated antibody vial before opening and using contents e The Stop Solution is an acidic solution e The kit sh
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