Isolation and Purification of Proteins
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.
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.
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