Quantification by Absorbance at 280nm

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This method is most accurate with highly purified proteins.

  1. Using nanodrop measure absorbance in triplicate, ensuring to blank against an appropriate buffer.
  2. Calculate extinction coefficient for protein of interest
  1. Use the Protparam tool to calculate the extinction coefficient in both molar and mg/mL for protein. Assume all Cys residues are half-cysteines.
  2. If the protein is a GST-fusion add 43110 M-1 and 1.607 mg/mL-1 respectively
  1. Divide the absorbance value (normalized by dilution factor if necessary) by the extinction coefficient
  2. If necessary adjust by the percent purity as described in Determining Percent Purity