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    • #17035
      scholesmu
      Participant

      i have no idea how the route of the calculation.

      1. 2.6 ml UV-assay buffer add to cuvette
      2. 50 ul sample add to curvette and measure absorbance 324 nm (A324 nm of sample)
      3. 200 ul DTDP solution add to curvette. If the absorbance is greater than 1, repeat 2 and 3 with less starting sample. Measure the absorbance at 324 nm. Note the absorbance (A324 of reaction)

      4. Calculation the protein concentration and cysteine concentration of the samples.

    • #112986
      JackBean
      Participant

      you need to know either extintion coeficient or have some calibration done

    • #112987
      jonmoulton
      Participant

      You will measure, dilute and measure until you get an absorbance between 1 and 2 (in the Beer’s law range). As Jack wrote, you need a molar extinction coefficient. Your path length is likely 1cm for a garden-variety spectrometer. Solve Beer’s law for the concentration, then plug in your values for the extinction coefficient, the path length and the absorbance.

      Solve Beer’s law for concentration and look at the units. Absorbance has no units. The units of the extinction coefficient are typically M^-1 cm^-1; in that case, a 1cm cell path length cancels out the cm^-1 unit and you’ll end up with a concentration in moles/liter. Watch the units on your extinction coefficient, sometimes they are different (you could, for instance, have an extinction coefficient calibrated as (pounds/quart)^-1 inches^-1, though anyone reporting an extinction coefficient in those units should be tarred and feathered).

      The concentration you get from Beer’s law is the concentration in the cell at the time the measurement was made. You need to calculate back to the concentration of the original sample in 50ul. That’s a pretty straightforward dilution calculation. You can use V1C1=V2C2.

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