Theories Behind The Methods
UV Spectrometer Theory:
Proteins and nucleic acids absorb light strongly in the ultraviolet region of the visible spectrum. The greatest absorption for proteins falls between the ranges of 200-280nm. The aromatic side chains of certain amino acids (phenylalanine, tyrosine, tryptophan) show an absorption in the area of 270-290 nm. In measuring protein concentration, the absorption reading of 280nm is typically used.
The theory behind UV spectroscopy and protein concentration is based on Beer’s Law (Al= el lc). By measuring the absorbance of the protein solution and determining the extinction coefficient (el) for a specific protein, the concentration of the protein can be determined.
The theory behind the Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) is to separate proteins based on size through the use of a stacking gel and resolving gel.
The samples that will be observed are loaded into the wells after being combined with a buffer containing 30% glycerol, SDS, and thiol. A voltage is then applied (approximately 3-4 v/cm2).
Since a higher charge and the small size of a molecule increases the mobility of that molecule in the stacking gel (pH=6.8), the resultant migration pattern of the molecules would be molecules with high charge and small size would be in the first row and in the last row would be the small charged large molecules. The rows in between would have the molecules organized by their mobility. The chloride ions in the stacking gel has the greatest mobility and highest charge. The proteins have a lower mobility than the chloride ions, but are faster than the glycine anions from the buffer. The resultant stack would be the chloride ions, proteins, and then the glycine anions.
After the icons stack in the stacking gel, they enter the resolving gel (pH=8.8), which has smaller pore sizes. The first event that occurs is that the higher pH places a greater negative charge on the small glycine anions. This results in the glycine anions migrating faster than the chloride ions. The second event is that the decrease in pore size creates a large frictional component on the mobility of each protein. Since the SDS in the gel creates a charge to mass ration that is equal, the proteins now migrate based on size.
The bands created by the proteins of different size can be compared to a standard that is run along with the sample. A standard curve can be created from the standard’s bands and the samples can then be compared to the standard.
Protein Assay Theory:
Four spectroscopic methods are routinely used to determine the concentration of protein in a solution. These include measurement of the protein's inherent 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. Although one or more these methods is used routinely in almost every biochemical laboratory, none of the procedures are particularly convenient
The Bio-Rad Protein Assay is based on the method of Bradford. Using the Bio-Rad protein assay is a simple and accurate procedure for determining concentration of solubilized protein. This procedure involves the addition of an acidic dye to a protein solution and then measuring absorbance at 595 nm with a spectrophotometer. By comparing the proteins absorbance curve to a standard curve, a relative measurement of protein concentration can be found.
The Bio-Rad Protein Assay is a dye-binding assay in which a differential color change of a dye occurs in response to various concentrations of protein. Absorbance max. for an acidic solution of Coomassie Brilliant Blue G-250 dye shifts from 465à595 nm when binding to protein occurs. Coomassie blue dye binds mainly to basic and aromatic amino acid residues, such as arginine. Accurate amounts of the protein are mandatory for this procedure, it is important to select an appropriate ratio of dye volume to sample concentration. Interferences for this procedure include chemical-protein and/ or chemical-dye interactions. It should be noted that for the Bio-Rad Protein Assay, basic buffer conditions and detergents do interfere with final results.
Bio-Rad Protein Assay. http://www.bio-rad.com/LifeScience/pdf/Bulletin_9004.pdf. USA.2-5.
Mathews, Christopher et al. Biochemistry 3rd edition. Addison Wesley Longman, Inc. ©2000. San Francisco, CA. 203-204.
Switzer, Robert. Experimental Biochemistry 3rd Edition. W.H. Freeman & Company. ©1999. New York, NY. 68-71.