Recoverin ELISA Kit

Catalog No. ABIN5564586

Product Details Recoverin ELISA Kit

Target: Recoverin (RCVRN)

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 0.313-20 ng/mL

Minimum Detection Limit: 0.313 ng/mL

Application: ELISA

Purpose: 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 immunoassay technique that measures human recoverin in approximately 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.

Brand: AssayMax™

Sample Type: Cell Culture Cells, Plasma, Serum, Tissue Lysate

Analytical Method: Quantitative

Components: Human Recoverin Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against human recoverin. Sealing Tapes: Each kit contains 3 precut, pressure sensitive sealing tapes that can be cut to fit the format of the individual assay. Human Recoverin Standard: Human recoverin in a buffered protein base (80 ng, lyophilized). Biotinylated Human Recoverin Antibody (50x): A 50-fold concentrated biotinylated polyclonal antibody against human recoverin (140 l). MIX Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (30 ml). Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml, 2 bottles). Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold concentrate (80 l). Chromogen Substrate: A ready-to-use stabilized peroxidase chromogen substrate tetramethylbenzidine (8 ml). Stop Solution: A 0.5 N hydrochloric acid to stop the chromogen substrate reaction (12 ml).

Material not included: Microplate reader capable of measuring absorbance at 405 nm. Pipettes (1-20 µL, 20-200 µL, and multiple channel). Deionized or distilled reagent grade water Incubator (37 °C)

Application Details

Plate: Pre-coated

Protocol:

• Step 1. Add 50 μL of Standard or Sample per well. Incubate 2 hours.
• Step 2. Wash, then add 50 μL of Biotinylated Antibody per well. Incubate 2 hours.
• Step 3. Wash, then add 50 μL of SP Conjugate per well. Incubate 30 minutes.
• Step 4. Wash, then add 50 μL of Chromogen Substrate per well. Incubate 15 minutes.
• Step 5. Add 50 μL of Stop Solution per well. Read at 450 nm immediately.

Reagent Preparation:

Freshly dilute all reagents and bring all reagents to room temperature before use. MIX Diluent Concentrate (10x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the MIX Diluent Concentrate 10-fold with reagent grade water. Store for up to 30 days at 2-8 °C. Human Recoverin Standard: 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 from the standard stock solution (20 ng/mL) 2-fold 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 stock solution should be frozen at -20 °C and used within 30 days. Avoid repeated freeze-thaw cycles. 4 Standard Point Dilution [Human Recoverin] (ng/mL) P1 1 part Standard (20 ng/mL) 20.0 P2 1 part P1 + 1 part MIX Diluent 10.0 P3 1 part P2 + 1 part MIX Diluent 5.0 P4 1 part P3 + 1 part MIX Diluent 2.5 P5 1 part P4 + 1 part MIX Diluent 1.25 P6 1 part P5 + 1 part MIX Diluent 0.625 P7 1 part P6 + 1 part MIX Diluent 0.313 P8 MIX Diluent 0.0 Biotinylated Human Recoverin Antibody (50x): Spin down the antibody briefly and dilute the desired amount of the antibody 50-fold with MIX Diluent. The undiluted antibody should be stored at -20 °C. Wash Buffer Concentrate (20x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the Wash Buffer Concentrate 20-fold with reagent grade water. SP Conjugate (100x): Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 100-fold with MIX Diluent. The undiluted conjugate should be stored at -20 °C.

Sample Collection: 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 collect plasma. Samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Serum: Samples should be collected into a serum separator tube. After clot formation, centrifuge samples at 3000 x g for 10 minutes and remove serum. Samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Tissue: Extract tissue samples with 50 mM phosphate-buffered saline ( pH 7.4) containing 1 % Triton X-100. Centrifuge at 14000 x g for 20 minutes. Collect the supernatant and measure the protein concentration. User should determine optimal dilution factor. Samples can be stored at -20 ° C or below. Avoid repeated freeze-thaw cycles. Cell Culture Supernatants: Centrifuge cell culture media at 3000 x g for 10 minutes to remove debris and collect supernatants. Samples can be stored at -20 ° C or below. Avoid repeated freeze-thaw cycles.

Assay Procedure:

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). 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. Add 50 l of Human Recoverin Standard or sample per well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Cover wells with a sealing tape and incubate for 2 hours. Start the timer after the last addition. Wash five times with 200 l 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 l of Wash Buffer and then invert the plate, decanting the contents, hit 4-5 times on absorbent material to completely remove the liquid. Add 50 l of Biotinylated Human Recoverin Antibody to each well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Cover wells with a sealing tape and incubate for 2 hours. Wash the microplate as described above. 5 Add 50 l of Streptavidin-Peroxidase Conjugate to each well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Cover wells with a sealing tape 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 l of Chromogen Substrate per well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Incubate for 15 minutes or till the optimal blue color density develops. Add 50 l of Stop Solution to each well. The color will change from blue to yellow. Gently tap plate to ensure thorough mixing. Break any bubbles that may have formed. 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.

Calculation of Results:

• 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.

Restrictions: For Research Use only

Handling

Handling Advice: This product is for Research Use Only and is not intended for use in diagnostic procedures. Prepare all reagents (diluent buffer, wash buffer, standard, biotinylated antibody, and SP conjugate) as instructed, prior to running the assay. 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. Spin down the SP conjugate vial and the biotinylated antibody vial before opening and using contents. The Stop Solution is an acidic solution. The kit should not be used beyond the expiration date. 2

Storage: 4 °C,-20 °C

Storage Comment: Upon arrival, immediately store components of the kit at recommended temperatures up to the expiration date. Store SP Conjugate and Biotinylated Antibody at -20°C. Store Microplate, Diluent Concentrate (10x), Wash Buffer, Stop Solution, and Chromogen Substrate at 2-8°C. 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. Diluent (1x) may be stored for up to 30 days at 2-8°C. Store Standard at 2-8°C before reconstituting with Diluent and at -20°C after reconstituting with Diluent.

Target Details for Recoverin

Target: Recoverin (RCVRN)

Alternative Name: Recoverin (RCVRN Products)

Synonyms: RCV1 ELISA Kit, CAR ELISA Kit, S-modulin ELISA Kit, Rcv1 ELISA Kit, rcv1 ELISA Kit, rcvrn ELISA Kit, recoverin ELISA Kit, reco ELISA Kit, recoverin ELISA Kit, recoverin, gene 2 S homeolog ELISA Kit, recoverin, gene 2 L homeolog ELISA Kit, Recoverin ELISA Kit, RCVRN ELISA Kit, Rcvrn ELISA Kit, rcvrn.2.S ELISA Kit, rcvrn.2.L ELISA Kit, reco ELISA Kit

Background: 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).

Gene ID: 5957

UniProt: P35243

Pathways: Regulation of G-Protein Coupled Receptor Protein Signaling, Phototransduction