Myoglobin ELISA Kit

Catalog No. ABIN5564582

Product Details Myoglobin ELISA Kit

Target: Myoglobin (MB)

Reactivity: Mouse

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 1.25-80 ng/mL

Minimum Detection Limit: 1.25 ng/mL

Application: ELISA

Purpose: The AssayMax™ Mouse Myoglobin ELISA (Enzyme-Linked Immunosorbent Assay) Kit is designed for detection of myoglobin in mouse plasma, serum, urine, tissue extracts, and cell culture samples. This assay employs a quantitative sandwich enzyme immunoassay technique that measures mouse myoglobin in approximately 4 hours. A polyclonal antibody specific for mouse myoglobin has been pre-coated onto a 96-well microplate with removable strips. Myoglobin in standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for mouse myoglobin, which is recognized by a streptavidin-peroxidase (SP) 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, Urine

Analytical Method: Quantitative

Components: Mouse Myoglobin Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against mouse myoglobin. Sealing Tapes: Each kit contains 3 precut, pressure sensitive sealing tapes that can be cut to fit the format of the individual assay. Mouse Myoglobin Standard: Mouse myoglobin in a buffered protein base (960 ng, lyophilized). Biotinylated Mouse Myoglobin Antibody (50x): A 50-fold concentrated biotinylated polyclonal antibody against mouse myoglobin (120 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). SP Conjugate (100x): A 100-fold concentrate (80 l). Chromogen Substrate (1x): A stabilized peroxidase chromogen substrate tetramethylbenzidine (8 ml). Stop Solution (1x): A 0.5 N hydrochloric acid solution 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

Assay Time: 5 h

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 1 hour.
• 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 to produce a 1x solution. Store for up to 30 days at 2-8 °C. Mouse Myoglobin Standard: Reconstitute the Mouse Myoglobin Standard (960 ng) with 3 mL of MIX Diluent to generate a 320 ng/mL standard stock solution. Allow the vial to sit for 10 minutes with gentle agitation prior to making dilutions. From the standard stock solution (320 ng/mL), dilute 4-fold with MIX Diluent to produce an 80 ng/mL standard working solution. Prepare duplicate or triplicate standard points by serially diluting the standard working solution (80 ng/mL) 2-fold with equal volume of MIX Diluent to produce 40, 20, 10, 5, 2.5, and 1.25 ng/mL solutions. MIX Diluent serves as the zero standard (0 ng/mL). Any remaining stock solution should be stored at -20 °C and used within 30 days. Avoid repeated freeze-thaw cycles. 5 Standard Point Dilution [Mouse Myoglobin] (ng/mL) P1 1 part Standard (320 ng/mL) + 3 parts MIX Diluent 80 P2 1 part P1 + 1 part MIX Diluent 40 P3 1 part P2 + 1 part MIX Diluent 20 P4 1 part P3 + 1 part MIX Diluent 10 P5 1 part P4 + 1 part MIX Diluent 5.0 P6 1 part P5 + 1 part MIX Diluent 2.5 P7 1 part P6 + 1 part MIX Diluent 1.25 P8 MIX Diluent 0.0 Biotinylated Mouse Myoglobin Antibody (50x): Spin down the antibody briefly and dilute the desired amount of the antibody 50-fold with MIX Diluent to produce a 1x solution. 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 to produce a 1x solution. SP Conjugate (100x): Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 100-fold with MIX Diluent to produce a 1x solution. 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. The sample is suggested for use at 1x, however, user should determine optimal dilution factor depending on application needs. If necessary, dilute samples within the range of 1x - 5x with MIX Diluent. The undiluted samples can be stored 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). 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. The sample is suggested for use at 1x, however, user should determine optimal dilution factor depending on application needs. If necessary, dilute samples within the range of 1x - 5x with MIX Diluent. The undiluted samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Urine: Collect urine using sample pot. Centrifuge samples at 800 x g for 10 minutes. The sample is suggested for use at 1x, however, user should determine optimal dilution factor depending on application needs. If necessary, dilute samples within the range of 1x - 5x with MIX Diluent. The undiluted samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Tissue Extracts: Extract tissue samples with 0.1 M phosphate-buffered saline ( pH 7.4) containing 1 % Triton X-100 and centrifuge at 14000 x g for 20 minutes. Collect the supernatant and measure the protein concentration. The remaining extract can be stored at -20 °C or below. Cell Culture Supernatants: Centrifuge cell culture media at 3000 x g for 10 minutes at 4 °C 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 Mouse Myoglobin Standard or sample 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. 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 Mouse Myoglobin Antibody to each well. Gently tap plate to thoroughly coat the wells. Break any bubbles that 6 may have formed. Cover wells with a sealing tape and incubate for 1 hour. Wash the microplate as described above. Add 50 l of SP 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 to each well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Incubate for 15 minutes or until 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. 2 The kit should not be used beyond the expiration date.

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 Myoglobin

Target: Myoglobin (MB)

Alternative Name: Myoglobin (MB Products)

Synonyms: PVALB ELISA Kit, AI325109 ELISA Kit, zgc:65819 ELISA Kit, zgc:77764 ELISA Kit, MB ELISA Kit, DKFZp468H096 ELISA Kit, myg ELISA Kit, mb ELISA Kit, MYF4 ELISA Kit, bHLHc3 ELISA Kit, myo ELISA Kit, Myoglobin ELISA Kit, myoglobin ELISA Kit, myogenin ELISA Kit, MB ELISA Kit, Mb ELISA Kit, mb ELISA Kit, myg ELISA Kit, Myog ELISA Kit

Background: Myoglobin is a heme-containing globular protein that is expressed in skeletal and cardiac muscles (1-2). Myoglobin consists of a single polypeptide chain of about 154 amino acid residues with a molecular weight of 17.6 kDa. It contributes oxygen storage and diffusion. Myoglobin also functions as a radical scavenger and prevents hypoxia. In the cardiovascular system, the myoglobin protein is abundantly expressed in the cytoplasm of cardiomyocytes and, to a much lesser extent, vascular smooth muscle (3). It has nitric oxide dioxygenation activity, which serves as a nitrite reductase and intracellular catalyst (4).

Gene ID: 17189

UniProt: P04247

Pathways: Brown Fat Cell Differentiation