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P a g e
Applications
Protein Determination
Protein Determination at 595, 546, 562 and 750 nm
The Bradford method depends on quantitating the binding of a dye, Coomassie Brilliant Blue, to an unknown protein
and comparing this binding to that of different, known concentrations of a standard protein at 595 nm; this is usually
BSA, bovine serum albumin.
The Biuret method depends on reaction between Cupric ions and peptide bonds in an alkali solution, resulting in the
formation of a complex absorbing at 546 nm.
The BCA method also depends on reaction between cupric ions and peptide bonds, but in addition combines this
reaction with the detection of cuprous ions using bicinchoninic acid (BCA), giving an absorbance maximum at 562 nm.
The BCA process is less sensitive to the presence of detergents used to break down cell walls.
The Lowry method depends on quantifying the colour obtained from the reaction of Folin-Ciocalteu phenol reagent
with the tylsryl residues of an unknown protein and comparing with those derived from a standard curve of a standard
protein at 750 nm; this is usually BSA, bovine serum albumin
Detailed protocols are supplied with these assay kits, and must be closely followed to ensure accurate results are
obtained.
The use of plastic disposable cells is recommended. To use a zero concentration standard include it in the number of
standards to be entered and enter 0.00 for concentration; use this when required to enter standard 1.
A linear regression analysis of the calibration standard data points is calculated; the result, together with the
correlation coefficient, can be printed out. A correlation coefficient of between 0.95 and 1.00 indicates a good straight
line.
