How to Calculate the Molarity of Proteins

Written by john brennan
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How to Calculate the Molarity of Proteins
Scientists often measure protein concentrations using a technique called the Bradford assay. (Hemera Technologies/ Images)

When purifying proteins, you need to be able to calculate their concentration. There are a variety of ways to do so, including the Biuret test, the Lowry assay, the BCA assay and the Bradford assay, all of which have advantages and disadvantages. In the end, however, they all provide essentially the same information: absorbance of a diluted sample versus absorbance at the same wavelength for solutions of standard concentration. When you have this data, you can use it to calculate your protein concentration.

Skill level:

Things you need

  • Pencil
  • Paper
  • Calculator
  • Spreadsheet program

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  1. 1

    Log into your spreadsheet program and enter the data for the standards in a new spreadsheet. Put "molarity" of the proteins in the standard solution in one column and "absorbance" at the wavelength you used in another.

  2. 2

    Graph the standards data and label your graph. Make sure molarity is on the x axis and absorbance is on the y.

  3. 3

    Using the linear regression or trend line tools in your spreadsheet program, find a line of best fit for your data. If necessary, consult your spreadsheet program's user manual for instructions.

  4. 4

    Take the absorbance for your unknown concentration protein solutions and plug them into the equation derived by your spreadsheet program. You can use your spreadsheet program to do the calculations for you if it will be helpful.

    Example: If your spreadsheet program derived the equation y = 0.5 x + 0.4, where y is absorbance and x is molarity, you can solve this equation for x by rearranging it as follows:

    (y - 0.4) / 0.5 = x

    then plug in your absorbance measurement for y as follows:

    (0.5 - 0.4) / 0.5 = x

    0.1 / .5 = 0.2 moles per litre

Tips and warnings

  • Note that protein concentration is typically calculated in terms of micrograms per microliter rather than moles per litre, although you can use molarity instead if you prefer.

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