Quantitation of protein.

TLDR: This chapter discusses various methods of estimating protein concentration as defined by the difference in energy between the orbital of the unexcited electron and a higher energy orbital.

Abstract: Publisher Summary This chapter discusses various methods of estimating protein concentration. Absorption spectroscopy involves the absorption of a photon by an electron. Only those photons with a certain energy level can be absorbed as defined by the difference in energy between the orbital of the unexcited electron and a higher energy orbital. The peptide bond absorbs photons below 210 nm. Because of the large number of peptide bonds in a protein, this is a highly sensitive area of the protein spectrum. Although protein conformation and some absorption by tryptophan and tyrosine residues occurs in this region, less variability between proteins is observed than at 280 nm. There is a need to avoid storing buffers in plastic containers because some plastics leach plasticizers, which absorb at ultraviolet (UV) wavelengths. Detergents can also be troublesome because many absorb UV light. If the buffer or protein solution is cold, the outside of the cuvette may need to be wiped between each reading with a lint-free wiper and the readings should be made quickly after placing the cold solution into the cuvette, because atmospheric moisture may condense on the outside of the cuvette producing an erroneously high reading.