Determination of protein concentration is an essential technique in all aspects of protein studies and proteomics. The Assays for Protein Quantification kit includes three of the most widely used protein assays and allows for a direct comparison of the three assays that teaches students the benefits and limitations of each assay. Each assay is available individually to allow teaching of a specific assay, without the option of comparing and contrasting with other assays. The three assays covered are the Biuret Protein Assay, Lowry Protein Assay and the Coomassie Blue Dye Protein Assay. The Assays for Protein Quantification kit provides all the reagents required to perform both protein assays, including protein standards for accurate quantification, in a single lab activity. An often underestimated factor in quantifying protein is the presence of non-protein interfering agents, such as salts and detergents. This kit teaches students about common laboratory agents that affect the protein assays, the reasoning behind their interferences and how to overcome the interference. Students also learn how to select a protein assay for different applications.
- Three protein assays or as individual assays
- Teaches three widely used methods of estimation: Biuret, Lowry and Coomassie Dye Protein Assays
- Understand effects of common laboratory agents on protein estimation
Under alkaline conditions cupric ions (Cu2+) chelate with the peptide bonds resulting in reduction of cupric ions (Cu2+) to cuprous ions (Cu+). The Cuprous ions can be detected with Folin Ciocalteu Reagent (phosphomolybdic/phosphotungstic acid); this method is commonly referred to as the Lowry method. Reduction of Folin Ciocalteu Reagent by cuprous ions (Cu+) produces a blue colour that can be read at 650-750 nm. The amount of colour produced is proportional to the amount of peptide bonds.
The Coomassie Blue Dye Protein Assay is based on the binding of protein molecules to Coomassie dye under acidic conditions. The binding of protein to the dye results in a spectral shift, the colour of Coomassie solution changes from brown (absorbance maximum 465 nm) to blue (absorbance maximum 610 nm). The change in colour density is read at 595 nm and is proportional to the protein concentration.
The Biuret assay is a copper ion based protein assay, protein solutions are mixed with an alkaline solution of copper salt, cupric ions (Cu2+). The protein assay is based on the interaction of cupric ions with protein in an alkaline solution. The interaction of cupric ions (Cu2+) with protein results in a purple color that can be read at 545 nm. The amount of colour produced is proportional to protein concentration.
Also required: Spectrophotometer and cuvettes or microplate reader and microplate.
Leveringsinformation: Supplied with components needed for hands-on experimentation for six workstations of 4-5 students or 24-30 students. Supplied with Teacher’s Guide and separate Student’s Guides.