AssayMax Human Recoverin ELISA Kit
Contents
1. 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 The undiluted samples can be stored at 20 C or below for up to 3 months Avoid repeated freeze thaw cycles 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 The undiluted samples can be stored at 20 C or below for up to 3 months Avoid repeated freeze thaw cycles e Tissue Extract tissue samples with 50 mM phosphate buffered saline pH7 4 containing 1 Triton X 100 and centrifuge at 14000 x g for 20 minutes Collect the supernatant and measure the protein concentration User should determine optimal dilution factor The samples can be stored at 20 C or below for up to 3 months Avoid repeated freeze thaw cycles e Cell Culture Supernatants Centrifuge cell culture media at 3000 x g for 10 minutes to remove debris Collect supernatants and assay The 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 _ MIX Diluent Concentrate 10x If crystals have formed in the concentr2. blue color density develops Gently tap plate to ensure thorough mixing and break the bubbles in the well with pipette tip Add 50 ul of Stop Solution to each well The color will change from blue to yellow 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 Calculate the mean value of the duplicate or triplicate readings for each standard and sample To generate a standard curve plot 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 Determine the unknown sample concentration from the Standard Curve and multiply the value by the dilution factor Standard Curve The curve is provided for illustration only A standard curve should be generated each time the assay is performed H Recoverin Standard Curve OD 450 nm 0 1F 0 1 1 0 10 0 100 0 h Recoverin ng ml Performance Characteristics e The minimum detectable dose of recoverin is typically 0 3 ng ml e Intr
3. A assarpro AssayMax Human Recoverin 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 15 minutes Step 5 Add 50 ul of Stop Solution per well Read at 450 nm immediately Symbol Key 3 Consult instructions for use Assay Template 12 11 10 Human Recoverin ELISA Kit Catalog No ER5010 1 Sample insert for reference use only Introduction Recoverin is a calcium dependent inhibitor of rhodopsin kinase a member of the EF hand family of calcium binding proteins involved in the transduction of light by vertebrate photoreceptors 1 3 Principle of the Assay The AssayMax Human Recoverin ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of human recoverin in plasma serum tissue extracts and cell culture samples This assay employs a quantitative sandwich enzyme
4. a assay and inter assay coefficients of variation were 5 0 and 7 1 respectively Linearity Average Percentage of Expected Value Sample Dilution Plasma Serum No Dilution 88 94 1 2 98 98 1 4 105 105 Recovery Standard Added Value 0 6 10 ng ml Recovery 83 110 Average Recovery 96 9796 Cross Reactivity Species Cross Reactivity Bovine None Mouse None Rat None Swine 80 Monkey None Canine None Rabbit None References 1 Higgins MK et al 2006 J Biol Chem 14 281 28 19426 32 2 Arthur S et al 1995 Proc Natl Acad Sci Vol 92 pp 9176 9180 3 Bazhin AV et al 2007 Cancer Immunol Immunother Jan 56 1 110 6 Version 1 0R www assaypro com e e mail Support assaypro com 1 SEP 2014
5. ate 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 Standard Curve Reconstitute the 80 ng of Human Recoverin Standard with 4 ml of MIX Diluent to generate a 20 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 20 ng ml 1 2 with MIX Diluent to produce 10 5 2 5 1 25 0 625 and 0 313 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 ae Human Recoverin Dilution Point ng ml P1 1 part Standard 20 ng ml 20 00 1 part P1 1 part MIX Diluent 10 00 1 part P2 1 part MIX Diluent 5 000 P4 ipartP3 1parMiXDiluent 250 P6 1partPS 1partMixDiluen 065 P8 MiDiuent ooo e Biotinylated Human Recoverin Antibody 50x Spin down the 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 desi
6. immunoassay technique that measures human recoverin in less than 5 hours A polyclonal antibody specific for human recoverin has been pre coated onto a 96 well microplate with removable strips Recoverin in standards and samples is sandwiched by the immobilized polyclonal antibody and biotinylated polyclonal antibody specific for human recoverin 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 should not be used beyond the expiration date Reagents e Human Recoverin Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against human recoverin e Sealing Tapes Each kit contains 3 precut pressure sensitive sealing tapes that can be cut
7. red 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 Human Recoverin 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 Human Recoverin 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 Wash the microplate as described above Add 50 ul of Chromogen Substrate per well and incubate for 15 minutes or till the optimal
8. to fit the format of the individual assay e Human Recoverin Standard Human recoverin in a buffered protein base 80 ng lyophilized e Biotinylated Human Recoverin Antibody 50x A 50 fold concentrated biotinylated polyclonal antibody against human recoverin 140 ul e _ MIX 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 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
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