(Phenol Red Binding to Serum Albumin)
Introduction: Serum albumin is often known as blood albumin, it is the most abundant plasma protein in humans and other mammals. Serum albumin is essential for maintaining the osmotic pressure that is needed for distribution of body fluids between body tissues and intravascular compartments. It is also a carrier protein by non specifically binding several biomolecules. 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.
In this experiment, we used the phenol red to bind to the albumin. Phenol red …show more content…
Sephadex G-25 is a cross-linked dextran polymer that has an exclusion limit approximately 30,000. Sephadex beads are porous, molecules larger than 30,000 are easily separated from molecules because it cannot enter the pores and are elute first. The molecules less than the exclusion limit will elute slower because it enter the pores and are retard (#2 …show more content…
In this part, we will see the correlation of the absorbance to concentrations and the thickness of the material used (cuvette). This is also stated in Beer-Lambert’s Law (A=εlc). A stands for Absorbance, ε is absorptivity, and l is the length of solution the light passes through, c is a concentration of solution. Absorbance is directly proportional to concentration. The more color of the sample, the more concentration. According to Ninfa, Ballou and Benore, the binding of phenol red to serum albumin is pH sensitive with a binding maximum in the range of pH 3.0 to 5.0 theoretically ((#3 Ninfa, Ballou, Benore). Based on theory, pH 4 that we used in this experiment should be optimum pH for albumin and phenyl red binding.
Materials:
• A packed gel- chromatography column with sephadex G-25
• 0.1 M Potassium Phosphate buffer at pH 6
• 0.1 M Sodium acetate buffer at pH