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Insertion and Isolation of Plasmids

Protein Purification

Determining Protein Concentration

SDS Gel Electrophoresis

BLAST

Conclusion

References

DETERMINING PROTEIN CONCENTRATION

Four spectroscopic methods are routinely used to determine the concentration of protein in a solution.  These include measurement of the protein's intrinsic UV absorbance and three methods which generate a protein-dependent color change; the Lowry assay, the Smith copper/bicinchoninic assay and the Bradford dye assay.  This investigation uses UV absorbance and the Bradford dye assay. 

UV Absorbance

Theory: There are many molecules that absorb ultraviolet or visible light.  Absorbance is related to the light path and the concentration of the sample by Beer’s Law, A=εbc, where b is the path length, c is the concentration of the absorbing species, and ε is the constant of proportionality also known as absorptivity. The absorbance increases as the concentration increases. The absorbance of these molecules occurs at different wavelengths. Molecules can be distinguished from one another by looking at an absorption spectrum. The spectrum is useful because it displays the number of absorption bands that corresponds to each structural group of the molecule.
The instrument used to make spectroscopic measurements is the spectrophotometer.  A light source sends beams of light through a monochromator. Next, the incident light (I_o) is sent through a cuvette containing the sample of interest. This is represented by I, the light path. Light that is not absorbed by the sample (I) can be detected. (See Figure 1) This method along with the Bradford dye assay is useful in determining the concentration of many different molecules.
 

Bio-Rad Protein Assay of an Unknown Concentration of pGlo Protein

Theory:  The Bradford dye assay is based on the equilibrium between three forms of Coomassie Blue G dye.  Under strongly acid conditions, the dye is most stable as a doubly-protonated red form.  Upon binding to basic and aromatic amino acid residues of proteins, however, it is most stable as an unprotonated, blue form.
 

The Bradford assay is fast, involves fewer mixing steps, does not require heating, and gives a more stable colorimetric response than the assays described above.  Like the other assays, however, its response is prone to influence from non-protein sources, particularly detergents, and becomes progressively more nonlinear at the high end of its useful protein concentration range.  The response is also protein dependent, and varies with the composition of the protein. These limitations make protein standard solutions necessary.