Isolation and Purification of Proteins

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The Isolation, purification and identification of Proteins assayed From Bovine Liver Using SDS Gel Electrophoresis, Mass Spectroscopy and Western Blotting

Abstract
The purpose of the experiment was to isolate and recognize varying protein solubilization and assaying methods by using bovine liver protein. The experiment implicated the impact of different types of solvents like ethanol, water, PBS, PBS+1% Triton x-100, and PBS+2% SDS on protein solubilization. Bradford and Ghosh/Dumbroff methods were used to calculate the amount of the dissolved proteins in the solvent. SDS-PAGE electrophoresis was used to separate the polypeptides in the mixture and was visualized by coomassie brilliant blue and silver staining. Western blot method was used to detect the molecular weight of proteins which is selected for three different antibodies: α-β-actin targeting the α and β isoforms of actin protein, α-GADPH targeting Glyceraldehyde 3-phosphate dehydrogenase, (GAPDH); and α-BSA which targets bovine liver protein. Target protein was identified with TANDEM mass spectrometry. The experiment can be labeled a success since it fulfilled its primary goal of isolation and identification of protein and also provided an appropriate comparison of Bradford and Ghosh/Dumbroff methods. The techniques employed in the experiment are fundamental aspects in the field of proteomics. Introduction

Amino acids are the basic building blocks of proteins that are joined by peptide bonds to form a peptide chain. These chains then conform to a special arrangement and interact with other peptide chains forming complex macromolecules known as proteins (Horton, et al., 1996). The amino acid content of a particular protein of interest can be determined and extracted through solubilization using detergents (Carpentier, et al., 2005). Solubilization of proteins is made possible by detergents due to the amphipathic behavior of the latter. A detergent has a hydrophilic head and a hydrophobic tail. The head is composed of a polar carboxylate group whereas the hydrophobic tail is a long alkyl chain (Schmid, 1996). Detergents exist in water as micelles, which are spherical clusters of hundreds of carboxylate ions dispersed throughout the water phase (Schmid, 1996). The hydrophilic portion faces outwards while the lipohilic tails point towards the center of the micelle. This behavior is similar to the lipid bilayer that forms the cell membrane, giving detergents the ability to dissolve proteins (Carpentier et al., 2005). Examples of detergents are PBS, PBS+1% Triton x-100, water, ethanol, and PBS+2% SDS which may be utilized in protein solubilization, thus facilitating identification and isolation of protein contents (Carpentier et al., 2005). Solubilization of proteins is made possible by detergents due to the amphipathic behavior of the latter. A detergent has a hydrophilic head and a hydrophobic tail. The head is composed of a polar carboxylate group whereas the hydrophobic tail is a long alkyl chain (Schmid, 1996). Detergents exist in water as micelles, which are spherical clusters of hundreds of carboxylate ions dispersed throughout the water phase (Schmid, 1996). The hydrophilic portion faces outwards while the lipohilic tails point towards the center of the micelle. This behavior is similar to the lipid bilayer that forms the cell membrane, giving detergents the ability to dissolve proteins (Carpentier et al., 2005). Examples of detergents are PBS, PBS+1% Triton x-100, water, ethanol, and PBS+2% SDS which may be utilized in protein solubilization, thus facilitating identification and isolation of protein contents (Carpentier et al., 2005). Peptide bonds can be broken down by ionic detergents like sodium dodecyl sulfate (SDS), which has effectively been used in membrane proteins (Laemmli, 1970). SDS has the ability to denature proteins into smaller fragments, which are then subjected to polyacrylamide gel electrophoresis (PAGE), allowing...
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