BL4820 Biochemistry Techniques -- Lecture 1 -- Protein Assay
3. Protein Assays
How do you determine the amount of protein in an unknown solution?
First, let's discuss what a protein is. Proteins are polymers of amino acids, where the amino acid units are joined by peptide bonds:
Structure of a Protein - Only the first 2 amino acid units of the protein are shown.
Peptide bonds have partial double bond character and absorb light at 220 nm in the far UV. Proteins also often contain the amino acids with aromatic rings in the side chains (ie. Trp, Tyr & Phe) which absorb at about 280 nm in the near UV. The presence of protein therefore can be detected using its UV absorbance: all proteins can be detected by absorbance at 220 nm (but the solution must be free of other UV absorbing substances - for example in the output stream of an HPLC where the background from the buffer or mobile phase can be subtracted from the protein peaks in the output) or by absorbance at 280 nm if the protein contains an aromatic amino acid.
The absorbance at 280 nm (A-280 nm) is most useful for detecting proteins in biochemical expts. In 1934, it was shown that for a mixture of yeast proteins, the A-280 nm for a 1 mg/ml solution was 1.0. This is a useful fact to remember and can be applied often to estimate the amount of protein present in a solution. You will use this in Expt #3 for estimating the amount of protein in the fractions you obtain when purifying the GOT enzyme.