Human HSP60 ELISA Kit
Contents
1. Recovery Standard Added Value 5 40 ng ml Recovery 96 91 110 Average Recovery 96 9696 Cross Reactivity Species Cross Reactivity 96 Bovine None Human 100 Mouse None Rat None Swine None Canine None Rabbit None Monkey None 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 Improper wash step e Check that the microplate washer is dispensing properly e If washing by pipette check for proper pipetting technique Low Precision Splashing of reagents while loading wells e Pipette properly in a controlled and careful manner Inconsistent volumes loaded into wells e Pipette properly in a controlled and careful manner e Check pipette calibration e Check pipette for proper performance Insufficient mixing of reagent dilutions e Thoroughly agitate the lyophilized components after reconstitution e Thoroughly mix dilutions Improperly sealed microplate e Check the microplate pouch for proper sealing e Check that the microplate pouch has no punctures e Check that three desiccants are inside the microplate pouch prior to sealing Unexpectedly Low or High Signal Intensity M2. 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 should not be used beyond the expiration date Reagents e Human HSP60 Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against human HSP60 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 Human HSP60 Standard Human HSP60 in a buffered protein base 80 ng lyophilized 2 vials e Biotinylated Human HSP60 Antibody 80x A 80 fold concentrated biotinylated polyclonal antibody against HSP60 75 ul e EIA Diluent Concentrate 10x A 10 fold concentrated buffered protein base 20 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 concentrated 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 Chromog
3. A assarbno AssayMax Human HSP60 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 Human Heat Shock Protein 60 HSP60 ELISA Kit Catalog No EH5505 1 Sample insert for reference use only Introduction Heat shock protein of 60 kDa HSP60 is a mitochondrial chaperonin involved in folding assembly and transport of newly imported protein from cytoplasm into mitochondria in an ATP mediated reaction 1 3 Human HSP60 contains 573 amino acids and is related to the bacteria groEL protein HSP60 is located in the mitochondria and cytoplasm the cell surface the extracellular space and the peripheral blood 4 5 Under dehydration condit
4. cing the assay in the incubator or at room temperature Improper pipetting e Pipette properly in a controlled and careful manner e Check pipette calibration e Check pipette for proper performance Insufficient mixing of reagent dilutions e Thoroughly agitate the lyophilized components after reconstitution e Thoroughly mix dilutions References 1 2 3 4 5 6 7 8 Cheng MY et al 1990 Nature 348 455 458 Goloubinoff P et al 1989 Nature 342 884 889 Ostermann J et al 1989 Nature 341 125 130 Reading DS et al 1989 Nature 337 655 659 Cappello F et al 2008 Cancer Biol Ther 7 801 809 Itoh H et al 2002 Eur J Biochem 269 5931 5938 Kim SC et al 2009 Circ Res 105 1186 1195 Magen D et al 2008 Am J Hum Genet 83 1 30 42 Version 1 4R Related Products EH5001 1 AssayMax Human Heat Shock Protein 27 HSP27 ELISA Kit Plasma Serum Milk Cell Culture Lysates and Tissue Extract samples EH5202 1 AssayMax Human Heat Shock Protein 47 HSP47 ELISA Kit Plasma Serum Milk Cell Culture Lysates and Tissue Extract samples www assaypro com e e mail Support assaypro com
5. en 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 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 Fresh standard should be reconstituted the day the assay is run 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 Store samples at 20 C or below for up to 3 months Avoid repeated freeze thaw cycles EDTA or 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 Collect the sample and assay Store samples at 20 C or below for up to 3 months Avoid repeated freeze thaw cycles e Cell Culture Lysates Place the cell culture dish in ice and wash the cells with ice cold PBS Drain the PBS then add ice cold lysis buffer 20 mM Tris HCl pH 7 5 150 mM NaCl 1 mM Na2EDTA 1 mM EGTA 1 Triton 0 1 mM PMSF 1 ug ml leupeptin 1 g mL aprotinin and 1 ug mL pepstatin Scrape adhere
6. he 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 Human HSP60 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 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 p
7. icroplate was left unattended between steps e Each step of the procedure should be performed uninterrupted Omission of step e Consult the provided procedure for complete list of steps Steps performed in incorrect order e Consult the provided procedure for the correct order Insufficient amount of reagents added to wells e Check pipette calibration e Check pipette for proper performance Wash step was skipped e Consult the provided procedure for all wash steps Improper wash buffer e Check that the correct wash buffer is being used Improper reagent preparation e Consult reagent preparation section for the correct dilutions of all reagents Insufficient or prolonged incubation periods e Consult the provided procedure for correct incubation time Deficient Standard Curve Fit Non optimal sample dilution e Sandwich ELISA If samples generate OD values higher than the highest standard point P1 dilute samples further and repeat the assay e Competitive ELISA If samples generate OD values lower than the highest standard point P1 dilute samples further and repeat the assay User should determine the optimal dilution factor for samples Contamination of reagents e A new tip must be used for each addition of different samples or reagents during the assay procedure Contents of wells evaporate e Verify that the sealing film is firmly in place before pla
8. ions the cytoplasmic HSP60 is quickly imported into the mitochondria by cytoplasmic HSP70 6 Extracellular HSP60 mediates apoptosis via Toll like receptors 7 An HSP60 defect can cause neurodegenerative pathologies 8 Principle of the Assay The AssayMax Human Heat Shock Protein 60 HSP60 ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of human HSP60 in plasma serum cell culture lysates and tissue samples This assay employs a quantitative sandwich enzyme immunoassay technique that measures human HSP60 in less than 5 hours A polyclonal antibody specific for human HSP60 has been pre coated onto a 96 well microplate with removable strips HSP60 in standards and samples is sandwiched by the immobilized antibody and biotinylated polyclonal antibody specific for HSP60 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
9. lot the graph using the standard concentrations 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 P1 Average OD 80 00 P2 40 00 P3 20 00 P4 10 00 PS 5 000 P6 2 500 P7 0 000 Sample Normal Sodium Citrate Plasma 1x Standard Curve e The curve is provided for illustration only A standard curve should be generated each time the assay is performed OD 450 nm Human HSP60 Standard Curve 0 1 1 A L rl 0 10 0 100 0 hHSP60 ng ml Performance Characteristics e The minimum detectable dose of HSP60 as calculated by 25D from the mean of a zero standard was established to be 2 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
10. nt cells off the dish and transfer the cell suspension into a pre cooled microfuge tube Maintain constant agitation for 30 minutes at 4 C Centrifuge in a microcentrifuge at 4 C Collect fresh cell lysates The undiluted samples can be stored at 20 C or below e Tissue Extract tissue samples with 50 mM phosphate buffered saline pH7 4 containing 196 Triton X 100 and centrifuge at 14000 x g for 20 minutes Collect the supernatant and measure the protein concentration The undiluted samples can be stored at 20 C or below 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 80 ng of Human HSP60 Standard with 1 ml of EIA Diluent to generate an 80 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 80 ng ml 1 2 with EIA Diluent to produce 40 20 10 5 and 2 5 ng ml solutions EIA Diluent serves as the zero standard 0 ng ml Any remaining solution should be discarded Fresh standard should be reconstituted the day the assay is run Standard Point Dilu
11. tion HSP60 ng ml P1 1 part Standard 80 ng ml 80 00 1 part P1 1 part EIA Diluent 40 00 1 part P2 1 part ElA Diluent 20 00 Pa 1partP3 1 part EIA Diluent 100 P6 ipartPS ipartFlADiluent 250 e Biotinylated Human HSP60 Antibody 80x Spin down the antibody briefly and dilute the desired amount of the antibody 1 80 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 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 Human HSP60 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 t
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