Spectrophotometric determination of protein concentration.
The concentration of a purified protein in solution is most conveniently and accurately measured using absorbance spectroscopy. The absorbance, A, is a linear function of the molar concentration, C, according to the Beer-Lambert law: A = epsilon x l x c, where e is the molar absorption coefficient and l is the cell path length. This unit provides protocols for calculation of epsilon for a folded or unfolded protein, making use of the average epsilon values for the three contributing chromophores in proteins (the side chains of Trp, Tyr, and Cys). A basic protocol describes how to measure the concentration of a protein using the calculated epsilon and the Beer-Lambert law. A sensitive method is provided for measuring the concentration of proteins that contain few if any tryptophan or tyrosine residues, and a simple method is provided for estimating total protein concentration in crude extracts.
Grimsley
Gerald R
GR
The Texas A&M University System Health Science Center, College Station, Texas.
Pace
C Nick
CN
The Texas A&M University System Health Science Center, College Station, Texas.
D017510:Protein Folding; D011506:Proteins / Q000032:analysis*; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*
D011506:Proteins