BL483 Biochemistry Techniques -- Lecture 1 -- Protein Assay
4. Commonly Used Protein Assay Methods
The common methods for determining protein depend on using a standard protein. There is
no absolute method for determining the protein concentration when you have a mixture of
proteins in a solution (see part 5 for discussion of pure proteins). So in general, a
specific protein which is readily available in quite pure form and not too expensive; for
example many biochemical labs use bovine serum albumin (BSA). In the expt this week in the
lab, you will use both BSA and the enzyme lysozyme, which is one of the major proteins in
egg white. By using a commonly available purified protein, it is easy for you and others
to reproduce the results you obtain.
The following methods are in wide spread use in biochemistry:
| Method |
How it Works |
Sensitivity Range |
| Biuret |
Cu2+-Peptide Bond Complex |
1 to 10 mg Protein |
| Folin (Lowry) |
Heavy Metal Complex with Aromatic Amino Acids |
20 to 300 µg Protein |
| Bradford (Bio-Rad) |
Dye Reaction with Amino Group Side Chains (Lys) |
1 to 100 µg Protein |
Description of Methods:
- Biuret - This method is the most linear because its color depends on a
direct complex between the peptide bonds of the protein and Cu(2+) ion. It is not highly
sensitive since the complex does not have a high extinction coefficient. You will use this
method to learn making a standard curve - your Biuret Standard Curve should be nearly
perfectly linear!
- Folin (Lowry) - The Folin assay (also called Lowry method after the
scientist who discovered it and published a description of the method in 1951) is
dependent on the presence of aromatic amino acids in the protein. First, a cupric/peptide
bond complex is formed and then this is enhanced by a phosphomolybodate complex with the
aromatic amino acids. Overall, about 10 to 50 times more sensitive than the Biuret method.
The Folin Standard Curve is usually not perfectly linear, but in your expt in this class
your results are usually quite linear if the assay is properly done. Many substances
interfere with the Folin assay for protein.
- Bradford assay is based on a blue dye (Coomassie Brilliant Blue) that binds to free amino
groups in the side chains of amino acids, especially Lys. This assay is as sensitive as
the Folin assay and can be done so that it is more sensitive, especially using the
commercial kits available from Bio-Rad and some other chemical companies. This method can
yield quite linear standard curves with BSA but often is found to be not quite linear. Few
substances interfere with this assay. The assay can also be done in the presence of
detergent which makes it useful for determining protein concentrations for membrane
proteins.
Summary - There is no universal assay for protein mixtures. It can be expected that
new methods will be developed in the future.
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