Implen P 300 User Manual

NanoPhotometer
®
P-Class User Manual
Version 2.1 Page 19 / 70
4.2 Protein Determination
4.2.1 General Information
Protein determination at 280 nm (NanoVolume Applications and Cuvette Applications)
 Protein can be determined in the near UV 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 A280 factor for a particular protein must be determined.
 The protein concentration can be calculated the following way:
c
prot.
= Abs. 280 * A280 factor * lid factor * dilution factor
With background correction:
c
prot.
= (Abs. 280 – Abs. 320) * A280 factor * lid factor * dilution factor
This equation can be applied to other proteins if the corresponding factors are known (please note that the factor
used by the NanoPhotometer
®
P-Class is the reciprocal value of the extinction coefficient (l/g*cm) from a protein).
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.
 The A280 Factor is based on the extinction coefficient of the protein [molecular weight/molar extinction coefficient
(M
-1
*cm
-1
) or 1/extinction coefficient (l/g*cm)].
In the software are the following protein A280 factors pre-programmed:
BSA (bovine serum albumin), serum albumin (mouse and human), lysozyme, IgG and OD 1 for more information
about the factors see 11.3 Protein quantification.
There is also the possibility to enter custom factors. For correct calculation the following settings are needed, either
the extinction coefficient (l/g*cm) or the molar extinction coefficient (M
-1
*cm
-1
) and the molecular weight (g/mol)
of the protein.
 Rapid measurements such as this at 280 nm are particularly useful after isolation of proteins and peptides from
mixtures using spin and HiTrap columns by centrifuge and gravity, respectively.
Protein determination at 280 nm and degree of labelling (NanoVolume Applications and Cuvette Applications)
 To determine the degree of labelling, the absorbance reading at the wavelength reported for maximum absorbance
of the fluorescence dye is used. The corresponding extinction coefficient of the dye is used in the Lambert-Beer
Law to determine the dye concentration (c = A / (e * d)). Absorbance values and extinction coefficients are used to
calculate the dye per protein ratio. For further details please refer to 12.4 Protein fluorescent dye incorporation.
Colorimetric Bradford, Biuret, BCA and Lowry protein determination (Cuvette Applications)
 The Bradford method depends on quantifying 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 is based on the Biuret reaction. Under alkaline conditions the divalent copper ion forms a
complex with peptide bonds in which it is reduced to a monovalent ion. Monovalent copper ion and the radical
groups of tyrosine, tryptophan, and cysteine react with Folin reagent to produce an unstable product that becomes
reduced to molybdenum/tungsten blue. Bound reagent changes colour from yellow to blue. This binding is
compared with those derived from 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.
 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.

Implen P 300 Specifications

General

Brand	Implen
Model	P 300
Category	Measuring Instruments
Language	English