1.Protein Estimation
Protein determination is necessary to estimate the amount of protein
in the sample, to normalise against the protein concentration
or during purification procedures. Depending on the amount of
sample, accuracy and presence of interfering agents, one needs
to decide on the method to be used. For accurate quantification,
the sample protein is compared with a known amount of a standard
protein which could either be the commonly used bovine
serum albumin (BSA) or it could sometimes be immunoglobulin
G (IgG). The various methods and their specifications are outlined
below:
1.1 Absorbance Assays
The aromatic rings in the protein absorb ultraviolet light at an
absorbance maximum of 280 nm, whereas the peptide bonds
absorb at around 205 nm. The unique absorbance property of
proteins could be used to estimate the level of proteins. These
methods are fairly accuratewith the ranges from 20μg to 3mg for
absorbance at 280 nm, as compared with 1 to 100μg for 205 nm.
The assay is non-destructive as the protein in most cases is not
consumed and can be recovered. Secondary, tertiary and quaternary
structures all affect absorbance; therefore, factors such as
pH, ionic strength, etc can alter the absorbance spectrum. This
assay depends on the presence of amino acids which absorb UV
light (mainly tryptophan, but to a lesser extent also tyrosine).
Small peptides that do not contain such amino acids cannot be
measured easily by UV.
Requirements
- Quartz Cuvettes
- UV-Spectrophotometer