Quantification by Absorbance at 280nm
From Bridges Lab Protocols
This method is most accurate with highly purified proteins.
- Using nanodrop measure absorbance in triplicate, ensuring to blank against an appropriate buffer.
- Calculate extinction coefficient for protein of interest
- Use the Protparam tool to calculate the extinction coefficient in both molar and mg/mL for protein. Assume all Cys residues are half-cysteines.
- If the protein is a GST-fusion add 43110 M-1 and 1.607 mg/mL-1 respectively
- Divide the absorbance value (normalized by dilution factor if necessary) by the extinction coefficient
- If necessary adjust by the percent purity as described in Determining Percent Purity