Structural Biochemistry/Proteins/Purification

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General Information[edit]

Protein Purification is the process of separating proteins for individual analysis. Protein purification is the second step of studying proteins, the first being the process of an assay. An assay is a procedure to measure the activity enzyme activity thus confirming the presence of the protein or proteins in interest. Popular assays include Western Blotting and ELISA(Enzyme-linked immunosorbent assay). Before the purification process, Cell Disruption is utilized to homogenize the cell's content. After the cell has been opened up, the process of purifying proteins from one another and the other organelles can be approached in several different methods. Protein mixtures are normally separated multiple times, each based on a different property, such as:

  • Solubility
  • Size
  • Molecular Weight
  • Charge
  • Binding affinity

The intended reason for purifying a specific protein governs the level and degree of protein purification. At times, a sample of protein that is only moderately purified suffices for its intended application; however, other situations require a higher degree of purification, especially if the fundamental ambition is to study the characteristics and tendencies of the specific protein in interest. By considering solubility, size, molecular weight, charge, and binding affinity, the goal of the scientist that conducts protein purification is to find a level of purification necessary and create a protein yield that is ample for further research and application. This means using the fewest amount of steps in order to keep the yield high, as each protein purification step incurs a degree of product loss. Therefore two factors serve as obstacles in protein purification: yield and purification level. The main goal of each protein purification project falls under two categories: analytical (for studying and research purposes) and preparative (for production and creation of commercial products).


There are many methods of purification including:







Proteins Purification Methods

.

Differential Centrifugation Salting Out Gel-Filtration Chromatography Ion-Exchange Chromatography Affinity Chromatography Hydrophobic Interaction Chromatography Gel Electrophoresis Isoelectric Focusing Two-Dimensional Electrophoresis Dialysis
Proteins are separated based on masses or densities by a centrifugal force. Centrifugation enables the separation of proteins in different cell compartments. Different proteins precipitate at different salt concentration. When the concentration of salt increases, more proteins are able to separate. Large molecules flow more rapidly to the bottom of the column. Proteins are separated according to its charge. Positively charged proteins bind to negatively charge bead, and negatively charge proteins are released. The negatively charged proteins flow through faster. Many proteins have high affinity for specific chemical groups. Proteins separate according to different levels of hydrophobicity. Electrophoresis separate protein while the gel enhances the separation. Small proteins move more rapidly through the gel. Different proteins have different pI (isoelectric point). Proteins are separated horizontally based on pI and vertically based on mass Proteins are separated through a semi-permeable membrane. Since the dimensions of proteins are generally larger than the pores of the membrane, proteins do not pass through and separate.