Ovalbumin Essay

The purpose of this experiment is to learn how to prepare albumin from egg white and to understand the basics of protein fractionation using ammonium sulfate. Ovalbumin is the major protein of egg white and is fractionated by means of ammonium sulfate precipitation. The properties of the solvent system will dissolve the protein and greatly impact solubility. In total, there are four major factors that affect protein solubility: ionic strength, pH, temperature, and dielectric constant of solvents and these are extremely useful in the field of biochemistry as well as for this experiment. Ionic strength is where protein solubility is affected when other ions are present and “salting in” stabilizes the charged protein group. In pH, any change will affect the ionization of functional groups. Temperature affects protein solubility because as it increases, proteins unfold with the non-polar groups becoming exposed. Finally, dielectric constants affect protein solubility because organic solvents actually decrease the dielectric constant of aqueous solutions and ultimately make proteins less soluble. One of the main pieces of equipment we will be using to perform this experiment is the centrifuge which is used to separate biomolecules and cell components. Materials The main equipment we will be using are the centrifuge, spectrophotometer, cuvette, and double pan balance. The reagents we will use include eggs, acetic acid 1.0 N, saturated ammonium sulfate, cheesecloth, bio-rad color reagent, BSA standard 0.2mg/ml, phosphate buffered saline, and bio-rad desalting column. Methods JA 20 rotor: Begin by turning on the centrifuge thirty minutes before we begin to use it so that way the rotor can cool down. Set the temperature and turn on the brake. Then we will use the double pan balance to balance the suspension holding the centrifuge tubes. We will then place the balanced tubes on opposite sides of the rotor and close the rotor and rotor chamber. We will then set the speed and time and will remove the tubes at the end. We will begin the experiment by cracking open an egg, separating the egg white, and measuring the volume of clear egg white after we’ve collected it in a graduated cylinder. …show more content…
We will then take a glass rod and thoroughly stir the egg and after that we will add 0.1 volume of 1.0 M acetic acid. Using a cheesecloth we will then filter the albumin and save the filtrate. We will then add an equal volume of buffered, saturated ammonium sulfate to the filtrate being sure to thoroughly stir and we will will then wait half an hour. For 15 minutes at 5EC we will centrifuge the above suspension at 10,000 rpm and after this process we will see two phases in the tube (the bottom precipitate has ovomucin and ovaglobulin and the other phase that is yellowish contains ovalbumin). We will pour off the supernatant into a new centrifuge tube and we will discard the pellet. Next, we will add saturated ammonium sulfate by the drops until the turbidity persists to the supernatant. We will then leave the tube at room temperature for about 4 hours and we will then centrifuge again. After removing the supernatant we will resuspend it in 3.0ml PBS. Finally, we will measure the volume of ovalbumin. Removal of Salt: We will be practicing two ways of removing salts from protein samples. The first is called gel filtration which is known as