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NanoPhotometer

N120/NP80/N60/N50/C40 User Manual Version 4.3.1

PROTEIN ASSAYS

METHOD OVERVIEW

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

order

regression analysis of the calibration standard data points is calculated by the

NanoPhotometer

. A correlation coefficient (R

) 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.

Note: Biuret is only available for cuvette measurements (NP80 and C40).

 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.

 Lowry Assay

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. The bound reagent changes color from yellow to blue. This binding is compared with

that obtained with a standard protein at 750 nm; this is usually BSA (bovine serum albumin).

Note: Detailed protocols are customarily supplied with these assay kits, and must be closely

followed to ensure that accurate results are obtained.

Implen NanoPhotometer NP80 User Manual

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