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Vinculin ELISA Kit

VCL Reaktivität: Human Colorimetric Sandwich ELISA 1.56-100 ng/mL Plasma, Serum, Tissue Homogenate
Produktnummer ABIN456790
  • Target Alle Vinculin (VCL) ELISA Kits anzeigen
    Vinculin (VCL)
    Reaktivität
    • 4
    • 3
    • 2
    • 2
    • 2
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    Human
    Nachweismethode
    Colorimetric
    Methodentyp
    Sandwich ELISA
    Detektionsbereich
    1.56-100 ng/mL
    Untere Nachweisgrenze
    1.56 ng/mL
    Applikation
    ELISA
    Verwendungszweck
    This immunoassay kit allows for the in vitro quantitative determination of human vinculin,VCL concentrations in serum, plasma, tissue homogenates and other biological fluids.
    Proben
    Plasma, Serum, Tissue Homogenate
    Analytische Methode
    Quantitative
    Spezifität
    This assay recognizes recombinant and natural human VCL.
    Kreuzreaktivität (Details)
    No significant cross-reactivity or interference was observed.
    Sensitivität
    < 0.78 ng/mL
    The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest detectable concentration that could be differentiated from zero.
    Produktmerkmale
    Homo sapiens,Human,Vinculin,Metavinculin,VCL,MV
    Bestandteile
    Reagent (Quantity): Assay plate (1×20ml), Standard (2), Sample Diluent (1×20ml), Assay Diluent A (1×10ml), Assay Diluent B (1×10ml), Detection Reagent A (1×120 μl), Detection Reagent B (1×120μl), Wash Buffer(25 x concentrate) (1×30ml), Substrate (1×10ml), Stop Solution (1×10ml), Plate sealer for 96 wells (5), Instruction (1)
    Benötigtes Material
    Luminometer. Pipettes and pipette tips. EP tube Deionized or distilled water.
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  • Probenmenge
    100 μL
    Plattentyp
    Pre-coated
    Protokoll
    The microtiter plate provided in this kit has been pre-coated with an antibody specific to VCL. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for VCL and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB substrate solution is added to each well. Only those wells that contain VCL, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of VCL in the samples is then determined by comparing the O.D. of the samples to the standard curve.
    Aufbereitung der Reagenzien

    Bring all reagents to room temperature before use. Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 30 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 750 mL of Wash Buffer. Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution produces a stock solution of 100 ng/ml. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions (Making serial dilution in the wells directly is not permitted). The undiluted standard serves as the high standard (100 ng/ml). The Sample Diluent serves as the zero standard (0 ng/ml). ng/mL 100 50 25 12.5 6.25 3.12 1.56 0 Detection Reagent A and B - Dilute to the working concentration using Assay Diluent A and B (1:100), respectively.

    Probennahme
    Serum - Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at approximately 1000 × g. Remove serum and assay immediately or aliquot and store samples at -20C or -80C. Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 × g at 2 - 8C within 30 minutes of collection. Store samples at -20C or -80C. Avoid repeated freeze-thaw cycles. Tissue homogenates - The preparation of tissue homogenates will vary depending upon tissue type. For this assay, tissue was rinsed with 1X PBS to remove excess blood, homogenized in 20 mL of 1X PBS and stored overnight at ≤ -20C After two freeze-thaw cycles were performed to break the cell membranes, the homogenates were centrifuged for 5 minutes at 5000 x g. Remove the supernate and assay immediately or aliquot and store at ≤ -20 . C Other biological fluids - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20C or -80C. Avoid repeated freeze-thaw cycles. Note: Serum, plasma and tissue homogenates to be used within 7 days may be stored at 2-8 C, otherwise samples must stored at -20C (≤ 1 months) or -80C (≤ 2 months) to avoid loss of bioactivity and contamination. Avoid freeze-thaw cycles. When performing the assay slowly bring samples to room temperature.
    Testdurchführung

    Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37C directly.). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at 4C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments.
    1. Add 100 μl of Standard, Blank, or Sample per well. Cover with the Plate sealer. Incubate for 2 hours at 37C.
    2. Remove the liquid of each well, don’t wash.
    3. Add 100 μl of Detection Reagent A working solution to each well. Cover with the 4 Plate sealer. Incubate for 1 hour at 37C. Detection Reagent A working solution may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.
    4. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (approximately 400 μl) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
    5. Add 100 μl of Detection Reagent B working solution to each well. Cover with a new Plate sealer. Incubate for 1 hours at 37C.
    6. Repeat the aspiration/wash five times as in step
    4. 7. Add 90 μl of Substrate Solution to each well. Cover with a new Plate sealer. Incubate within 30 minutes at 37C. Protect from light.
    8. Add 50 μl of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
    9. Determine the optical density of each well at once, using a microplate reader set to 450 nm.
    Important Note:
    1. Absorbance is a function of the incubation time. Therefore, prior to starting the assay it is recommended that all reagents should be freshly prepared prior to use and all required strip-wells are secured in the microtiter frame. This will ensure equal elapsed time for each pipetting step, without interruption.
    2. Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction, and avoid foaming and mix gently until the crystals have completely dissolved. The reconstituted Standards can be used only once. This assay requires pipetting of small volumes. To minimize imprecision caused by pipetting, ensure that pipettors are calibrated. It is recommended to suck more than 10μl for once pipetting.
    3. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. Do not allow wells to sit uncovered for extended periods between incubation steps. Once reagents have been added to the well strips, DO NOT let the strips DRY at any time during the assay.
    4. For each step in the procedure, total dispensing time for addition of reagents to the assay plate should not exceed 10 minutes.
    5. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.
    6. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
    7. Duplication of all standards and specimens, although not required, is recommended.
    8. Substrate Solution is easily contaminated. Please protect it from light. 5

    Ergebnisberechnung

    Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the x-axis against the concentration on the y-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the VCL concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. It is recommended to use some related software to do this calculation, such as curve expert 13.0. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

    Beschränkungen
    Nur für Forschungszwecke einsetzbar
  • Handhabung
    1. The kit should not be used beyond the expiration date on the kit label.
    2. Do not mix or substitute reagents with those from other lots or sources.
    3. If samples generate values higher than the highest standard, further dilute the 3 samples and repeat the assay. Any variation in standard diluent, operator, pipetting technique, washing technique,incubation time or temperature, and kit age can cause variation in binding.
    4. This assay is designed to eliminate interference by soluble receptors, ligands, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.
    Lagerung
    4 °C/-20 °C
    Informationen zur Lagerung
    The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20 °C upon being received. The other reagents can be stored at 4 °C.
  • Target Alle Vinculin (VCL) ELISA Kits anzeigen
    Vinculin (VCL)
    Andere Bezeichnung
    VCL (VCL Produkte)
    Synonyme
    CMD1W ELISA Kit, CMH15 ELISA Kit, MV ELISA Kit, MVCL ELISA Kit, VCL ELISA Kit, im:6902790 ELISA Kit, hm:zehn2160 ELISA Kit, DDBDRAFT_0186762 ELISA Kit, DDBDRAFT_0232320 ELISA Kit, DDB_0186762 ELISA Kit, DDB_0232320 ELISA Kit, 9430097D22 ELISA Kit, AA571387 ELISA Kit, AI462105 ELISA Kit, AW545629 ELISA Kit, CG3299 ELISA Kit, CT11081 ELISA Kit, Dmel\\CG3299 ELISA Kit, Dmvincp ELISA Kit, EG:103B4.1 ELISA Kit, Vin ELISA Kit, Vin2EF ELISA Kit, vinc ELISA Kit, LOC100228062 ELISA Kit, vcl-A ELISA Kit, vinculin ELISA Kit, vinculin a ELISA Kit, vinculin A ELISA Kit, Vinculin ELISA Kit, vinculin S homeolog ELISA Kit, VCL ELISA Kit, vcla ELISA Kit, vinA ELISA Kit, Vcl ELISA Kit, Vinc ELISA Kit, CpipJ_CPIJ013873 ELISA Kit, Bm1_37935 ELISA Kit, vcl ELISA Kit, vcl.S ELISA Kit
    Hintergrund
    In mammalian cells, vinculin is a membrane-cytoskeletal protein in focal adhesion plaques that is involved in linkage of integrin adhesion molecules to the actin cytoskeleton. Its sequence is 20-30% homologous to alpha-catenin, which serves a similar function. Binding alternately to talin or alpha-actinin, vinculin's shape and consequently its binding properties are changed. The vinculin gene occurs as a single copy and apparently no close relative to take over functions in its absence. Its splice variant metavinculin (see below) also needs vinculin to heterodimerize and work in a dependent fashion. Vinculin is a 117 kDa cytoskeletal protein with 1066 amino acids. The protein contains an acidic N-terminal domain and a basic C-terminal domain separated by a proline-rich middle segment. Vinculin consists of a globular head domain that contains binding sites for talin and alpha-actinin as well as a tyrosine phosphorylation site, while the tail region contains binding sites for F-actin, paxillin and lipids.
    Pathways
    Cell-Cell Junction Organization, Maintenance of Protein Location, Signaling Events mediated by VEGFR1 and VEGFR2
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