Fibronectin ELISA Kit

Catalog No. ABIN612774

Product Details Fibronectin ELISA Kit

Target: Fibronectin

Reactivity: Rat

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 0.098-100 ng/mL

Minimum Detection Limit: 0.098 ng/mL

Application: ELISA

Purpose: The AssayMax Rat Fibronectin ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for detection of rat fibronectin in plasma, serum, urine, and cell culture samples. This assay employs a quantitative sandwich enzyme immunoassay technique that measures rat fibronectin in 4 hours. A polyclonal antibody specific for rat fibronectin has been pre-coated onto a 96- well microplate with removable strips. Rat fibronectin in standards and samples is sandwiched by the immobilized antibody and biotinylated polyclonal antibody specific for rat fibronectin, 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, Urine

Analytical Method: Quantitative

Components: Rat Fibronectin Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against rat fibronectin. Sealing Tapes: Each kit contains 3 precut, pressure sensitive sealing tapes, which can be cut to fit the format of the individual assay. Rat Fibronectin Standard: Rat fibronectin in a buffered protein base (1.2 μg, lyophilized). Biotinylated Rat Fibronectin Antibody (40x): A 40-fold concentrated biotinylated polyclonal antibody against rat fibronectin (150 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

Sample Volume: 50 μL

Assay Time: 4 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 30 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 1:10 with reagent grade water. Store for up to 30 days at 2-8 °C. 4

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. Dilute samples 1:16000 into MIX Diluent and assay. The undiluted 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. Dilute samples 1:16000 into MIX Diluent and assay. 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 and assay. Store samples at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Cell Culture Supernatants: Centrifuge cell culture media at 3000 x g for 10 minutes to remove debris. Collect supernatants and assay. Store samples at -20 °C or below. Avoid repeated freeze-thaw cycles. Refer to Sample Dilution Guidelines below for further instruction. Guidelines for Dilutions of 1:100 or Greater (for reference only, please follow the insert for specific dilution suggested) 1:100 1:10000 A) 4 μL sample: 396 μL buffer(100x) = 100 fold dilution Assuming the needed volume is less than or equal to 400 μL. A) 4 μL sample : 396 μL buffer (100x) B) 4 μL of A : 396 μL buffer (100x) = 10000 fold dilution Assuming the needed volume is less than or equal to 400 μL. 1:1000 1:100000 A) 4 μL sample : 396 μL buffer (100x) B) 24 μL of A : 216 μL buffer (10x) = 1000 fold dilution Assuming the needed volume is less than or equal to 240 μL. A) 4 μL sample : 396 μL buffer (100x) B) 4 μL of A : 396 μL buffer (100x) C) 24 μL of B : 216 μL buffer (10x) = 100000 fold dilution Assuming the needed volume is less than or equal to 240 μL.

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 Rat Fibronectin Standard or sample per well. Cover wells with a sealing tape and incubate for 2 hours. Start the timer after the last addition. 5 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 Rat Fibronectin Antibody to each well and incubate for 1 hour. Wash the microplate as described above. Add 50 l of Streptavidin-Peroxidase Conjugate per 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 l of Chromogen Substrate per well and incubate for 30 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 l 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.

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.

Assay Precision: Intra-assay and inter-assay coefficients of variation were 4.2 % and 7.1% respectively.

Restrictions: For Research Use only

Handling

Handling Advice: 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. 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.

References for Fibronectin ELISA Kit (ABIN612774)

Hsieh, Liu, Hung, Hung, Liu, Chuang, Chen, Wang, Shi, Hsu, Shiue, Yang: "Elucidation of the therapeutic role of mitochondrial biogenesis transducers NRF-1 in the regulation of renal fibrosis." in: Experimental cell research, (2016) (PubMed).

Yan, Fang, Yang, Li, Lu, Cheng: "Anti-diabetic nephropathy compounds from Cinnamomum cassia." in: Journal of ethnopharmacology, Vol. 165, pp. 141-7, (2015) (PubMed).

Target Details for Fibronectin

Target: Fibronectin

Alternative Name: Fibronectin (FN) (Fibronectin Products)

Synonyms: FN ELISA Kit, cig ELISA Kit, fibronectin ELISA Kit, finc ELISA Kit, lets ELISA Kit, msf ELISA Kit, CIG ELISA Kit, ED-B ELISA Kit, FINC ELISA Kit, FNZ ELISA Kit, GFND ELISA Kit, GFND2 ELISA Kit, LETS ELISA Kit, MSF ELISA Kit, E330027I09 ELISA Kit, Fn ELISA Kit, Fn-1 ELISA Kit, FIBNEC ELISA Kit, fn-1 ELISA Kit, fibronectin 1 ELISA Kit, FN1 ELISA Kit, fn1 ELISA Kit, Fn1 ELISA Kit

Background: Fibronectin (FN) is a major component of the extracellular matrix and blood plasma and is a specific ligand for several integrin adhesion receptors (1). FN plays an important role not only in cell adhesion (2) and wound healing (3), but also in hematopoiesis (4).

Gene ID: 25661

UniProt: P04937