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User Manual

ELK16J0127, Rabbit Phosphorylated Histone (γ-H2AX)

standards concentration (X)
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
Rabbit γ-H2AX ELISA Kit instruction
Intended use
This γ-H2AX ELISA kit is intended Laboratory for Research use only and is not for use in diagnostic or
therapeutic procedures.The Stop Solution changes the color from blue to yellow and the intensity of the
color is measured at 450 nm using a spectrophotometer. In order to measure the concentration of γ
-H2AX in the sample, this γ-H2AX ELISA Kit includes a set of calibration standards. The calibration
standards are assayed at the same time as the samples and allow the operator to produce a standard curve
of Optical Density versus γ-H2AX concentration. The concentration of γ-H2AX in the samples is then
determined by comparing the O.D. of the samples to the standard curve.
Sample collection and storages
Serum - Use a serum separator tube and allow samples to clot for 30 minutes before centrifugation for
10 minutes at approximately 3000×g. Remove serum and assay immediately or aliquot and store samples
at -20℃or -80℃.Avoid repeated freeze-thaw cycles.
Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 30 minutes
at 3000×g at 2-8℃within 30 minutes of collection. Store samples at -20℃or -80℃. Avoid repeated
freeze-thaw cycles.
Cell culture supernates and other biological fluids - Remove particulates by centrifugation and
assay immediately or aliquot and store samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles.
Note: The samples shoule be centrifugated dequately and no hemolysis or granule was
allowed.
Materials required but not supplied

  1. Standard microplate reader(450nm).
  2. Precision pipettes and Disposable pipette tips.
  3. 37 ℃incubator.
    Precautions
    6.0ml 3.0ml
    6.0ml 6.0ml 3.0ml 3.0ml
    membrane 2 2
    1 Standard (S0 ￫S5) concentration was followed by:0,7.5,15,30,60,120 ng/mL. 1
    20×wash solution:Dilute with Distilled or deionized water 1:20.
    Prepare all reagents before starting assay procedure. It is recommended that all Standards and
    Add standard: Set Standard wells, testing sample wells. Add standard 50μl to standard well.
    Add Sample: Add testing sample 10μl then add 40μl of Sample Diluent to testing sample wel.
    any remaining Wash Solution by aspirating or decanting. Invert the plate and blot it against clean paper towels.
    1. Add chromogen solution A 50μl and chromogen solution B 50μl to each well. Gently mix and incubate for 15 minutes at 37°C. Protect from light.
    2. Add 50μl Stop Solution to each well. The color in the wells should change from blue to yellow. If the color in the wells is green or the color change does not appear uniform, gently tap the plate to ensure thorough mixing.
      8 Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader within 15 minutes.
      Calculation of results
    3. This standard curve is used to determine the amount in an unknown sample. The standard curve is generated by plotting the average O.D. (450 nm) obtained for each of the six standard concentrations on the vertical (Y) axis versus the corresponding concentration on the horizontal (X) axis.
    4. First, calculate the mean O.D. value for each standard and sample. All O.D. values, are subtracted by the mean value of the zero standard before result interpretation. Construct the standard curve using graph paper or statistical software.
    5. To determine the amount in each sample, first locate the O.D. value on the Y-axis and extend a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the X-axis and read the corresponding concentration.
    6. Any variation in operator, pipetting and washing technique, incubation time or temperature, and kit age can cause variation in result. Each user should obtain their own standard curve.
    7. The sensitivity by this assay is 1 ng/mL.
    8. Standard curve.
      standards concentration (X)
      Storage: 2-8℃. Validity:six months.
      FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS! PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!