Column Chromatography is considered one of the main techniques of separation in addition to SDS-PAGE and gel electrophoresis. This method can be useful in both a small scale as in a laboratory experiment as well as in a larger scale at the industrial level. It is also applied in different fields ranging from biology and biochemistry to microbiology and medicine.
Column chromatography is also referred to as gel filtration or size exclusion chromatography among many other terminologies and it is used to separate molecules solely based on their size. The concept behind this technique is quite simple and effective to determine the molecular weight of some molecules, specifically proteins, and to separate the components of a mixture. The procedure …show more content…
Cytochrome c is a protein that has been implicated in the apoptosis system in cells and is known to have a molecular weight of about 12kDa. It is also consists of a single chain of 104 amino acids as its primary structure (3) and it is soluble in water or buffered solutions with pH around 7 which results in a clear dark pink solution. The tertiary structure of the protein is important in terms of the presence of a heme group that has a role in interactions with amino acids (4). As of what concerns hemoglobin, it is the protein responsible for the red color in blood cells and also the most important respiratory protein as it has an important role in the transport of oxygen to tissue cells and the return of carbon dioxide. The molecular weight of bovine hemoglobin is about 64.5 kDa. In terms of its structure, hemoglobin is a tetramer consisting of 2 pairs of polypeptide chains and 4 heme groups. It is also soluble in water and results in a red-brown solution (5).
The main purpose of this experiment is to do determine the molecular weight of bovine cytochrome c and bovine hemoglobin by making use of the gel filtration technique using Sephacryl beads. The goal is to create first a standard curve of log of molecular weight as a function of volume eluted necessary for the elution of two standards, blue Dextran and Vitamin B-12 mixed in one solution (6). Determining the volume eluted for both cytochrome c and hemoglobin would allow the determination of their molecular weight through the standard
Column chromatography is also referred to as gel filtration or size exclusion chromatography among many other terminologies and it is used to separate molecules solely based on their size. The concept behind this technique is quite simple and effective to determine the molecular weight of some molecules, specifically proteins, and to separate the components of a mixture. The procedure …show more content…
Cytochrome c is a protein that has been implicated in the apoptosis system in cells and is known to have a molecular weight of about 12kDa. It is also consists of a single chain of 104 amino acids as its primary structure (3) and it is soluble in water or buffered solutions with pH around 7 which results in a clear dark pink solution. The tertiary structure of the protein is important in terms of the presence of a heme group that has a role in interactions with amino acids (4). As of what concerns hemoglobin, it is the protein responsible for the red color in blood cells and also the most important respiratory protein as it has an important role in the transport of oxygen to tissue cells and the return of carbon dioxide. The molecular weight of bovine hemoglobin is about 64.5 kDa. In terms of its structure, hemoglobin is a tetramer consisting of 2 pairs of polypeptide chains and 4 heme groups. It is also soluble in water and results in a red-brown solution (5).
The main purpose of this experiment is to do determine the molecular weight of bovine cytochrome c and bovine hemoglobin by making use of the gel filtration technique using Sephacryl beads. The goal is to create first a standard curve of log of molecular weight as a function of volume eluted necessary for the elution of two standards, blue Dextran and Vitamin B-12 mixed in one solution (6). Determining the volume eluted for both cytochrome c and hemoglobin would allow the determination of their molecular weight through the standard