NanoPhotometer
®
NP80/N60/N50/C40
User Guide Version 3.1
61
detected out of the acceptable range an alert icon
is shown in the results/table area. A click
on the alert icon shows additional information. The ranges for acceptable ratio values can be
defined in preferences.
The ratio is calculated with or without background correction according to if the background
correction is activated during the measurements or not as follows:
Without background correction:
260/280 ratio =
A
260
A
280
With background correction:
260/280 ratio =
A
260
- A
BKG
A
280
- A
BKG
P
ROTEIN
A
SSAYS
M
ETHOD
O
VERVIEW
Protein concentration may be measured using colorimetric assays, in which certain reagents are
added to the protein solution to generate a colored product; either a protein-cupric ion chelate
as in the Biuret, Lowry, BCA assays or a protein-dye complex as in the Bradford assay. In these
colorimetric assays, the absorbance is measured in the visible range at the appropriate
wavelength for each assay and compared against a standard curve prepared by serial dilution
of a protein standard of known concentration. A linear, linear zero or 2
nd
order regression
analysis of the calibration standard data points is calculated by the NanoPhotometer
®
. A
correlation coefficient (R
2
) in the range of 0.95 to 1.00 indicates a good fit to a straight line.
Bradford Assay
Method depends on quantifying the binding of a dye, Coomassie Brilliant Blue, to an unknown
protein and comparing this binding to that of a standard curve prepared from a set of known
protein of known concentrations at 595 nm. This standard is usually BSA (bovine serum
albumin).
Biuret Assay
Method depends on a reaction between cupric ions and peptide bonds in an alkali solution,
resulting in the formation of a complex absorbing at 546 nm.
BCA Assay
Method depends on a reaction between cupric ions and peptide bonds coupled 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 solubilize
membranes.
