AssayMaxTM Rat Myoglobin ELISA Kit
Contents
1. before opening and using contents e The Stop Solution is an acidic solution e Thekit should not be used beyond the expiration date Reagents e RatMyoglobin Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against rat myoglobin 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 RatMyoglobin Standard Rat myoglobin in a buffered protein base 480 ng lyophilized e Biotinylated Rat Myoglobin Antibody 50x A 50 fold biotinylated polyclonal antibody against rat myoglobin 120 ul e MIX Diluent Concentrate 10x A 10 fold 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 wit2. up to 3 months Avoid repeated freeze thaw cycles e Tissue Extract tissue samples with 0 1 M phosphate buffered saline pH7 4 containing 196 Triton X 100 and centrifuge at 14000 x g for 20 minutes Collect the supernatant measure the protein concentration and assay Freeze remaining extract at 20 C or below e Cell Culture Supernatants Centrifuge cell culture media at 3000 x g for 10 minutes to remove debris Collect supernatants and assay Store samples at 20 C or below Avoid repeated freeze thaw cycles Reagent Preparation e Freshly dilute all reagents and bring all reagents to room temperature before use e MIX Diluent Concentrate 10x If crystals have formed in the concentrate mix gently until the crystals have completely dissolved Dilute the MIX Diluent Concentrate 1 10 with reagent grade water Store for up to 30 days at 2 8 C e RatMyoglobin Standard Reconstitute the 480 ng of Rat Myoglobin Standard with 1 ml of MIX Diluent to generate a 480 ng ml standard stock solution Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions Prepare duplicate or triplicate standard points by serially diluting the standard stock solution 480 ng ml 1 4 with MIX Diluent to generate 120 30 7 5 and 1 875 ng ml solutions MIX Diluent serves as the zero standard 0 ng ml Any remaining solution should be frozen at 20 C and used within 30 days Standard Dilution Rat Myoglobin Point ng ml P1
3. 1 part Standard 480 ng ml 480 0 1 part P1 3 parts MIX Diluent 120 0 Pa 1ipartP3 3partsMiXDiluent 7500 P6 gt MiXDiunt 0000 e Biotinylated Rat Myoglobin Antibody 50x Spin down the biotinylated antibody briefly and dilute the desired amount of the antibody 1 50 with MIX 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 MIX 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 performed 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 Rat Myoglobin 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 mater
4. DA SSAYPRO AssayMax Rat Myoglobin 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 1 hour 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 30 minutes Step 5 Add 50 ul of Stop Solution per well Read at 450 nm immediately Symbol Key ci Consult instructions for use Assay Template 12 11 10 Rat Myoglobin ELISA Kit Catalog No ERM8001 1 Sample insert for reference use only Introduction Myoglobin is aheme containing globular protein that is expressed in skeletal and cardiac muscles 1 2 Human myoglobin consists of a single polypeptide chain of about 154 amino acid residues with a molecular weight of 17 6 kDa It contributes to oxygen storage and diffusion and functions as a radical scavenger and prevents hypoxia In the cardiovascular system myoglobin protein is abundantly expressed in the cytoplasm of cardiomyocytes and to a much less
5. are inside the microplate pouch prior to sealing Microplate was left e Each step of the procedure should be performed T unattended between uninterrupted 5 steps n Omission of step e Consult the provided procedure for complete list of steps 5 Steps performed in e Consult the provided procedure for the correct order r incorrect order 5 z Insufficient amount of e Check pipette calibration 3 Pa reagents added to Check pipette for proper performance 2 9 wells zs Wash step was skipped e Consult the provided procedure for all wash steps E Improper wash buffer e Check that the correct wash buffer is being used 9 Improper reagent Consult reagent preparation section for the correct preparation dilutions of all reagents u Insufficient or e Consult the provided procedure for correct incubation gt prolonged incubation time periods e Sandwich ELISA If samples generate OD values higher than the highest standard point P1 dilute samples further and repeat the assay Non optimal sample e Competitive ELISA If samples generate OD values lower dilution than the highest standard point P1 dilute samples further and repeat the assay e User should determine the optimal dilution factor for samples Contamination of e A new tip must be used for each addition of different reagents samples or reagents during the assay procedure Contents of wells e Verify that the sealing film is firmly in place before placing evaporate the a
6. er extent in vascular smooth muscle 3 It has nitric oxide dioxygenation activity which serves as a nitrite reductase and intracellular catalyst 4 Principle of the Assay The AssayMax Rat Myoglobin ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of rat myoglobin in plasma serum urine tissue extract and cell culture samples This assay employs a quantitative sandwich enzyme immunoassay technique that measures rat myoglobin in 4 hours A polyclonal antibody specific for rat myoglobin has been pre coated onto a 96 well microplate with removable strips The rat myoglobin in standards and samples is sandwiched by the immobilized antibody and biotinylated polyclonal antibody specific for rat myoglobin 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 e 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
7. h the desiccant packs and resealed May be stored for up to 30 days in a vacuum desiccator e Diluent 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 Plasma Collect plasma using one tenth volume of 0 1 M sodium citrate as an anticoagulant Centrifuge samples at 3000 x g for 10 minutes and assay If necessary dilute samples within the range of 1x 5x The user should determine the optimal dilution factor The undiluted samples can be stored at 20 C or below for up to 3 months Avoid repeated freeze thaw cycles EDTA and Heparin can also be used as an anticoagulant e Serum Samples should be collected into a serum separator tube After clot formation centrifuge samples at 3000 x g for 10 minutes Remove serum and assay If necessary dilute samples within the range of 1x 5x The user should determine the optimal dilution factor The undiluted samples can be stored at 20 C or below for up to 3 months Avoid repeated freeze thaw cycles e Urine Collect urine using sample pot Centrifuge samples at 800 x g for 10 minutes and assay Samples can be stored at 20 C or below for
8. ial 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 Rat Myoglobin Antibody to each well and incubate for 1 hour e Wash the microplate as described above e Add 50 ul of Streptavidin Peroxidase Conjugate per 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 30 minutes or till the optimal color density develops Gently tap the 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 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
9. on the x axis and the corresponding mean 450 nm absorbance OD on the y axis The best fit line can be determined by regression analysis using log log or four parameter logistic curve fit e Determine the unknown sample concentration from the Standard Curve and multiply the value by the dilution factor Typical Data e The typical data is provided for reference only Individual laboratory means may vary from the values listed Variations between laboratories may be caused by technique differences Standard Point Average OD Sample Normal Sodium Citrate Rat Plasma 1x Standard Curve e The curve is provided for illustration only A standard curve should be generated each time the assay is performed Rat Myoglobin Standard Curve OD 450 nm 01 L L a 1 10 100 1000 Rat Myoglobin ng ml Performance Characteristics e The minimum detectable dose of rat myoglobin as calculated by 2SD from the mean of a zero standard was established to be 1 1 ng ml e Intra assay precision was determined by testing replicates of three plasma samples in one assay e Inter assay precision was determined by testing three plasma samples in twenty assays Intra Assay Precision Inter Assay Precision Sample 1 2 3 1 2 3 n 20 20 20 20 20 20 CV 96 Average CV 96 Recovery Standard Added Value 7 5 120 ng ml Recovery 96 85 11296 Average Recove
10. ry 96 96 Linearity e Serum samples were serially diluted to test for linearity Average Percentage of Expected Value Sample Dilution Serum No Dilution 95 1 2 100 1 4 105 Cross Reactivity Species Cross Reactivity Canine None Bovine None Monkey lt 20 Mouse None Rabbit None Rat 100 Swine lt 60 Human lt 60 Troubleshooting Issue Causes Course of Action Use of expired e Check the expiration date listed before use components e Do not interchange components from different lots e Check that the correct wash buffer is being used e Check that all wells are dry after aspiration c Improper wash step e Check that the microplate washer is dispensing properly 2 e If washing by pipette check for proper pipetting o technique L Splashing of reagents e Pipette properly in a controlled and careful manner a while loading wells 3 z 3 Ineondistent volumes e Pipette properly in a controlled and careful manner loaded into wells e Check pipette calibration e Check pipette for proper performance Insufficient mixing of e Thoroughly agitate the lyophilized components after reagent dilutions reconstitution e Thoroughly mix dilutions e Check the microplate pouch for proper sealing Improperly sealed e Check that the microplate pouch has no punctures microplate e Check that three desiccants
11. ssay in the incubator or at room temperature e Pipette properly in a controlled and careful manner Improper pipetting e Check pipette calibration Deficient Standard Curve Fit e Check pipette for proper performance e Thoroughly agitate the lyophilized components after reconstitution e Thoroughly mix dilutions Insufficient mixing of reagent dilutions References 1 Weller P et al 1984 EMBO J 3 439 446 2 Akaboshi E 1985 Gene 33 241 249 3 Wittenberg JB and Wittenberg BA 2003 J Exp Biol 206 Pt 12 2011 2020 4 Rahaman MM and Straub AC 2013 Redox Biology 1 405 410 Version 1 2R Related Products e EMM8001 1 AssayMax Mouse Myoglobin ELISA Kit Plasma Serum Urine Cell Culture samples www assaypro com e e mail Support assaypro com
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