Transferrin ELISA Kit

Produktnummer ABIN612779

Produktdetails Transferrin ELISA Kit

Target: Transferrin (TF)

Reaktivität: Ratte

Nachweismethode: Colorimetric

Methodentyp: Competition ELISA

Untere Nachweisgrenze: 0.15 µg/mL

Applikation: ELISA

Verwendungszweck: The AssayMax Rat Transferrin ELISA (Enzyme-Linked Immunosorbent Assay) kit employs a quantitative competitive enzyme immunoassay technique that measures rat plasma, and serum Transferrin.

Marke: AssayMax

Proben: Plasma

Analytische Methode: Quantitative

Bestandteile: Rat Transferrin Microplate: A 96 well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against rat Transferrin. Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes that can be cut to fit the format of the individual assay. Rat Transferrin Standard: Rat Transferrin in a buffered protein base (20 µg, lyophilized). Biotinylated Transferrin: 1 vial, lyophilized. 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, 1 bottle). 1 Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold concentrate (80µl). 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).

Benötigtes Material: Microplate reader capable of measuring absorbance at 450 nm. Pipettes (1-20 µL, 20-200 µL, 200-1000µLand multiple channel). Deionized or distilled reagent grade water.

Anwendungsinformationen

Probenmenge: 25 μL

Testdauer: < 3 h

Plattentyp: Pre-coated

Protokoll: A polyclonal antibody specific for rat Transferrin has been pre-coated onto a 96-well microplate with removable strips. Transferrin in standards and samples is competed with a biotinylated Transferrin sandwiched by the immobilized antibody and streptavidin-peroxidase conjugate. All unbound material is then washed away and a peroxidase enzyme substrate is added. The color development is stopped and the intensity of the color is measured.

Aufbereitung der Reagenzien:

Freshly dilute all reagents and bring all reagents to room temperature before use. If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. MIx Diluent Concentrate (10x): Dilute the MIx Diluent 1:10 with reagent grade water. Store for up to 1 month at 2-8°C. Standard Curve: Reconstitute the 20 g of Transferrin Standard with 2 ml of MIx Diluent to generate a Standard Solution of 10 g/ml. 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 Solution (10 g/ml) 1:2 with MIx Diluent to produce 5, 2.5, 1.25, 0.625, 0.313, and 0.156 g/ml solutions. MIx Diluent serves as the zero standard (0 g/ml). Any remaining solution should be frozen at -20°C. Standard Point Dilution [R. Transferrin] ( g/ml) Standard Solution (10 g/ml) P1 10.000 P2 1 part P1 + 1 part MIx Diluent 5.000 P3 1 part P2 + 1 part MIx Diluent 2.500 P4 1 part P3 + 1 part MIx Diluent 1.250 P5 1 part P4 + 1 part MIx Diluent 0.625 P6 1 part P5 + 1 part MIx Diluent 0.313 P7 1 part P6 + 1 part MIx Diluent 0.156 P8 MIx Diluent 0.000 Biotinylated Transferrin (4x): Dilute Biotinylated Transferrin with 4 ml MIx Diluent to produce a 4-fold stock solution, which should be further diluted 1:4 with MIx Diluent. Any remaining solution should be frozen at -20°C. Wash Buffer Concentrate (20x): Dilute the Wash Buffer Concentrate 1:20 with reagent grade water. 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.

Probennahme: Plasma: Collect plasma using one-tenth volume of 0.1 M sodium citrate as an anticoagulant. Centrifuge samples at 2000 x g for 10 minutes and assay. Dilute samples 1:3000 into 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 anticoagulant). Serum: Samples should be collected into a serum separator tube. After clot formation, centrifuge samples at 2000 x g for 10 minutes. Remove serum and assay. Dilute samples 1:3000 into MIx Diluent. The undiluted samples can be stored at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles.

Testdurchführung:

Prepare all reagents, working standards and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at room temperature (20 - 30 °C). Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccant inside. Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator. Add 25 µL of standard or sample per well, and immediately add 25 µL of Biotinylated Transferrin to each well (on top of the Standard or sample) and mix gently. Cover wells with a sealing tape and incubate for two hours. Start the timer after the last sample addition. Wash five times with 200 µL of Wash Buffer manually. Invert the plate each time and decant the contents, hit it 4-5 times on absorbent paper towel to completely remove the liquid. If using a machine wash six times with 300 µL of Wash Buffer and then invert the plate, decant the contents, hit it 4-5 times on absorbent paper towel to completely remove the liquid. Add 50 µL 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 µL 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. Add 50 µL 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. 3

Ergebnisberechnung:

Calculate the mean value of the 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 on the y-axis and draw a best fit curve through the points on the graph. Plotting the 4 parameters or linear graph may linearize the data and the best-fit line can be determined by regression analysis of the linear portion of the curve. 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.

Testpräzision: Intra-assay and inter-assay coefficients of variation were 4.5% and 7.0% respectively.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Handhabung: Prepare all reagents (working diluent buffer, wash buffer, standards, biotinylated-protein, 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 protocol. However, the user should determine the optimal dilution factor. Spin down the SP conjugate vial before opening and using contents. The kit should not be used beyond the expiration date.

Lagerung: 4 °C/-20 °C

Informationen zur Lagerung: Store components of the kit at 2-8°C or -20°C upon arrival up to the expiration date. Store SP Conjugate at -20°C Store Microplate, Diluent Concentrate (10x), Wash Buffer, Stop Solution, and Chromogen Substrate at 2-8°C Opened unused microplate wells may be returned to the foil pouch with the desiccant packs. Reseal along zip-seal. May be stored for up to 1 month in a vacuum desiccator. Diluent (1x) may be stored for up to 1 month at 2-8°C. Store Standard and Biotinylated Protein at 2-8°C before reconstituting with Diluent and at -20°C after reconstituting with Diluent.

Details zu Transferrin

Target: Transferrin (TF)

Andere Bezeichnung: Transferrin (TF Produkte)

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Hintergrund: Transferrin is a plasma protein that transports iron through the blood to the liver, spleen and bone marrow. Low Transferrin levels in plasma could be associated with anemia and chronic liver disease. On the other hand, high plasma Transferrin levels could indicate iron deficiency anemia.

Pathways: Transition Metal Ion Homeostasis