Annexin V ELISA Kit (Annexin A5)

Details for Product ANXA5 ELISA Kit No. ABIN5564556, Supplier: Log in to see
Antigen
  • anx
  • anx5
  • ANX V
  • anxa5
  • cb989
  • wu:fa98f06
  • wu:fj10f10
  • MGC89158
  • ANX5
  • ENX2
  • PP4
  • RPRGL3
  • Anx5
  • R74653
  • LC5
  • enx2
  • annexin A5
  • annexin A5b
  • Annexin A5
  • annexin A5 L homeolog
  • ANXA5
  • anxa5b
  • anxa5
  • Anxa5
  • anxa5.L
Reactivity
Human
Alternatives
Kits with alternative reactivity to:
18
16
15
14
14
8
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7
4
2
2
2
2
1
1
1
1
Method Type
Sandwich ELISA
Detection Range
0.25-16 ng/mL
Minimum Detection Limit
0.25 ng/mL
Application
ELISA
Options
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Purpose The AssayMax™ Human Annexin A5 ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for detection of annexin A5 in human plasma, serum, saliva, and cell culture samples. This assay employs a quantitative sandwich enzyme immunoassay technique that measures human annexin A5 in approximately 4 hours. A polyclonal antibody specific for human annexin A5 has been pre-coated onto a 96-well microplate with removable strips. Annexin A5 in standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for human annexin A5, 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, Saliva, Serum
Analytical Method Quantitative
Detection Method Colorimetric
Components Human Annexin A5 Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against human annexin A5. Sealing Tapes: Each kit contains 3 precut, pressure sensitive sealing tapes that can be cut to fit the format of the individual assay. Human Annexin A5 Standard: Human annexin A5 in a buffered protein base (192 ng, lyophilized, 2 vials). Biotinylated Human Annexin A5 Antibody (50x): A 50-fold biotinylated polyclonal antibody against human annexin A5 (120 l). EIA Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (20 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)
Background Annexin V (Annexin A5, ANXA5) is a calcium dependent phospholipid binding protein of the annexin group. Although little is known about the function of this protein, it has been proposed that calcium-specific adsorption of annexin A5 to phospholipid monolayers induces polymerization of the protein in the plane of adsorption, potentially exerting a biological effect on the membrane itself or upon membrane-bound proteins. Electron image analysis has shown that annexin A5 Molecules form trimers in a triskelion pattern on lipid monolayers (1). Annexin A5 also displays upregulation and has been shown to interact with kinase insert domain receptor and integrin beta 5 (2-4).
Gene ID 308
UniProt P08758
Pathways Apoptosis
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. EIA Diluent Concentrate (10x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the EIA Diluent Concentrate 10-fold with reagent grade water. Store for up to 30 days at 2-8 °C. Human Annexin A5 Standard: Reconstitute the Human Annexin A5 Standard (192 ng) with 1.5 mL of EIA Diluent to generate a 128 ng/mL standard stock solution. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. The standard stock solution (128 ng/mL) should be further diluted 4-fold with EIA Diluent to produce a 32 ng/mL standard working solution. Prepare duplicate or triplicate standard points by serially diluting the standard working solution (32 ng/mL) 2-fold with EIA Diluent to produce 16, 8, 4, 2, 1, and 0.5 ng/mL solutions. EIA Diluent serves as the zero standard (0 ng/mL). Aliquot remaining stock solution to limit repeated freeze-thaw cycles. This solution should be stored at -20 °C and used within 48 hours. 4 Standard Point Dilution [ANXA5] (ng/mL) P1 1 part Standard (128 ng/mL) + 3 parts EIA Diluent 32 P2 1 part P1 + 1 part EIA Diluent 16 P3 1 part P2 + 1 part EIA Diluent 8.0 P4 1 part P3 + 1 part EIA Diluent 4.0 P5 1 part P4 + 1 part EIA Diluent 2.0 P6 1 part P5 + 1 part EIA Diluent 1.0 P7 1 part P6 + 1 part EIA Diluent 0.5 P8 EIA Diluent 0.0 Biotinylated Human Annexin A5 Antibody (50x): Spin down the antibody briefly and dilute the desired amount of the antibody 50-fold with EIA 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 EIA 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. The sample is suggested for use at 1x, however, user should determine optimal dilution factor depending on application needs. 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. The undiluted samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Saliva: Collect saliva using sample tube. 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. The undiluted samples can be stored 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 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 Human Annexin A5 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 Human Annexin A5 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 1 hour. 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 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 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 Standard, 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.
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