Gel Filtration Chromatography Lab Report

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Observe Ligand-Protein Interaction using Gel Filtration Chromatography
(Phenol Red Binding to Bovine Serum Albumin)

Introduction: Serum albumin is often known as blood albumin, and is the most abundant protein in humans and other animal plasma. Serum albumin is also known as a carrier protein by non-specifically binding several biomolecules. It is essential for maintaining the osmotic pressure that is needed for body fluids distribution between body tissues and intravascular compartments. Ligand is a molecule can bind and make a complex with a biomolecule for biological purpose. Its binds those molecules with intermolecular force such as H-bonding, ionic bond, and Van der Waals forces (#1. Worldlibrary.org).
In this experiment, we used the
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This technique is widely used in biochemical laboratories, and usually used in separation of macromolecules such as proteins, nucleic acids, and carbohydrates, etc. In this particular lab, the technique of gel filtration will be used to separate the phenol red- bovine serum albumin complex (PR-BSA). Before working with a chromatography column to separate or isolate specific molecules, it requires understanding the theory behind the technique. Most of chromatography works based on the same general principle: a sample mixture in a mobile phase (buffer) passes over a selective matrix that called stationary phase. The stationary phase or the matrix inside the column is a composed cross-linked polymer bead with engineered pores or cavities (#2 Lehninger, 87). In a well-packed column, the large molecule (larger than the largest pore) cannot enter the pores in the Sephadex beads. Thus, they will move around the beads and rapidly move through the column and elute first. Smaller molecules flow easily into the cavities to different degrees depends on their size; therefore they will take a longer path through the column. The elution buffer, or mobile phase, flows through the bed and out the column carrying the dissolved solutes with it, and it also keeps the bed from drying …show more content…
After the separation of molecules based on their size using gel filtration chromatography. The spectroscopic method was used to get the absorbance. Absorbance measurements used to quantify the ligand protein complex and free ligand concentrations. This part is based on the Beer-Lambert’s Law (A=εlc). A stands for Absorbance, ε is extinction coefficient or molar absorptivity (M-1 cm-1), and l is the length (thickness) that light passes through the sample (cm), c is a concentration of solution (M). Absorbance is directly proportional to concentration. The more color of the sample, the more concentration of the

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