Wittrup: Determining Protein Concentration
Determine protein concentration by UV/Vis spectrophotomtery
1. Concentrate protein solution
2. Determine extinction coefficient of protein. (Please refer to Gill and von Hippel, Calculation of protein extinction coefficients from amino acid sequence data, Analytical Biochemistry, (1989), 182, 319-326.
3. Transfer a small aliquot of the protein solution into known volume of 6M guanidine hydrochloride solution.
4. Transfer 6 M guanidine hydrochloride solution into a clean quartz cuvette. Use the cuvette to set the baseline of the spectrophotometer.
5. Empty, wash, and dry the cuvette.
6. Fill the cuvette with the protein solution. Measure the absorbance at an appropriate wavelength (usually 280 nm).
7. Determine the concentration using Beer’s law. That is concentration = A / eL in which e is the extinction coefficient and L is the path length of the cuvette.
To obtain reliable reading, the absorbance of the protein solution should be between 0.1 to 1.0