This application note gives a complete solution for determining the dye concentration, the protein concentration and the dye-to-protein ratio using a METTLER TOLEDO UV Vis spectrophotometer.
What Are Proteins?
Proteins are large, complex molecules performing vital roles in the body. They do most of the work in cells and are required for the structure, function, and regulation of the body’s tissues and organs. Proteins are composed of hundreds or thousands of repeating units known as amino acids, which are connected to each another in long chains. There are 20 different types of amino acids that can be combined to make a protein.
Role of Proteins
The sequence of amino acids determines each protein’s unique 3-dimensional structure and its specific function. On the basis of their myriad functional properties, proteins are classified as
- Antibody: protects the body against bacteria and viruses
- Enzymes: catalyzes several chemical reactions
- Messenger: hormonal
- Structural: provides structure and support to cells
- Storage: carry atoms and molecules
Quantification of Protein
Proteins can be effectively quantified by UV VIS when labelled with fluorescent dyes by measuring its absorbance. The dye utilized for labelling shall be stoichiometric to protein. Excess dye must be removed as this can cause fluorescent signal quenching or the deactivation of the protein assay. Nevertheless, optimal signal sensitivity should be provided to allow accurate concentration measurement. Hence, the quantification of the dye-to-protein ratio or the labelling ratio is crucial.
The concentration of the dye, the protein and the dye-to-protein ratio of the labelled protein can be determined using UV Vis spectroscopy .
UV5Nano which is a micro volume spectrophotometer is ideal for life science laboratories.
After measuring the absorbance of the labelled protein standard solutions, UV Vis micro-volume spectrophotometer automatically calculates the dye concentration, the protein concentration and the dye-to-protein ratio.
Download our free application note to learn more and benefit from our experience in determining protein content.
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