Hoefer SP-2001 Vision Life Spectrophotometer - Manual (Page 35)

Version 1.0
Page 35
Protein Determination
Protein Determination at 280 nm
• Protein can be determined at 280 nm due to absorption by tyrosine, tryptophan and phenylalanine amino acids;
Abs 280 varies greatly for different proteins due to their amino acid content, and consequently the specific
absorption value for a particular protein must be determined.
• The presence of nucleic acid in the protein solution can have a significant effect due to strong nucleotide
absorbance at 280 nm. This can be compensated by measuring Abs 260, and applying the equation of
Christian and Warburg for the protein crystalline yeast enolase (Biochemische Zeitung 310, 384 (1941)):
Protein (mg/ml) = 1.55 * Abs 280 - 0.76 * Abs 260
or, Protein conc. = (Factor 1 * Abs 280) - (Factor 2 * Abs 260)
• This equation can be applied to other proteins if the corresponding factors are known. The instrument can
determine protein concentration at 280 nm and uses the above equation as default; the factors can be
changed, and the use of background correction at 320 nm is optional.
• To customize the equation for a particular protein, the absorbance values at 260 and 280 nm should be
determined at known protein concentrations to generate simple simultaneous equations; solving these provides
the two coefficients. In cases where Factor 2 is found to be negative, it should be set to zero since it means
there is no contribution to the protein concentration due to absorbance at 260 nm.
• Set Factor 2 = 0.00 for direct λ280 UV protein measurement; Factor 1 is based on the extinction coefficient of
the protein. If BSA (bovine serum albumin) is an acceptable standard, setting Factor 1 = 1.115 will give linear
results from 0 to 0.8 mg/ml protein.
Protein (mg/ml) = 1.115 * Abs 280
• Rapid measurements such as this at Abs 280 are particularly useful after isolation of proteins and peptides
from mixtures using spin and HiTrap columns by centrifuge and gravity, respectively.
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 color obtained from the reaction of Folin-Ciocalteu phenol
reagent with the tyrosyl 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.