Electrophoresis Separation of Proteins Cytochrome C Myoglobin Hemoglobin and Serum Albumin by Using Isoelectric Focusing System IEF essays

Electrophoresis Separation of Proteins Cytochrome C Myoglobin Hemoglobin and Serum Albumin by Using Isoelectric Focusing System IEF essays Electrophoresis Separation of Proteins Cytochrome C, Myoglobin, Hemoglobin, and Serum Albumin by Using Isoelectric Focusing System (IEF) Proteins are composed of amino acids. All amino acids are amphoteric molecules consisting of three types of amino acids: neutral, acidic, and basic. Thus, for any protein there is a characteristic pH, called the isoelectric point (pI), at which the protein has no net charge and therefore will not move in the electric field. Electrophoresis takes advantage of this characteristic. Proteins are electrophoreased, and the most negatively charged protein moves closest to the cathode, and the most positively charged protein moves closest to the anode. Cytochrome C was expected to move closest to the cathode, and serum albumin was expected to move closest to the anode. Only cytochrome C was expected to move to the cathode. The other three proteins were expected to move toward anode. The purpose of electrophoresis was to see how a difference in pI makes a difference in the electrophoretic mobility of protein. Four proteins were electrophoreased by using the Tris-Glysin buffer of pH 8.6 and a horizontal agarose gel 1.1 % in isoelectric focusing (IEF) at a voltage of 175 V and at a current of 79 mA. The agarose gel was made by mixing 0.18g of agarose in 1.5ml of Tris-Glysin buffer with a pH of 8.6. That is 100 % * 0.18 (0.18 + 15) = 1.1% of agarose gel. 15 ƒÊl of each protein sample was loaded into each sample application well on the agarose gel without mixing with glycerol solution. After the agarose gels were placed on the stage of the electrophoresis chamber, Tris-Glysin buffer of pH 8.6 was filled in the electrophoresis chamber carefully until the agarose gels were slightly covered with the buffer. Four proteins had electrophoreased for about 50 minutes. The agarose gels were removed from the electrophoresis chamber and stained overnight with the Coomassie Blue to visualize proteins in the agarose gel....