Electrophoresis is the term used to describe the movement of ions under the influence of an electric field. In this field, cations and ions will both migrate towards the oppositely charged electrodes. Anions, naturally migrate towards the positively charged electrode and Cations on the other hand migrate towards the negatively charged electrode. Large molecules like protein can be separated by the process of electrophoresis.
Proteins are amphoteric. They can lose protons in base solutions to form negative ions. The more acidic R-group residues that are on a protein, the greater the negative charge on a protein in alkaline buffers. As a result, the more rapidly the protein fraction will migrate on a solid medium toward the positive…show more content… The staining can be done on the native plate or can be deferred until chemical or enzymatic reactions produce a stainable compound. Some stains are actually fluorescent and are visualised under ultra-violet light in a darkened room. Visual inspection is used to identify and crudely quantify bonds on a plate. Accurate quantitation requires densitometric measurements.
6Densitometer: This is a special type of spectrophotometer and is used to measure the light transmittance through a solid sample such as an electrophoresis plate. The plate is positioned over or in front of the exit slit of the monochromator. Most densitometers can scan a plate. This is usually accomplished by marking the plate across the exit slit.
General Procedure for Electrophoresis
Cellulose acetate medium is soaked in Barbitol buffer (pH 8.6). After blotting the medium, patients’ samples and controls are applied. A cellulose acetate strip is placed in an electrophoretic chamber and a current is applied. After a fixed period of time, the Cellulose Acetate film is removed and stained with Ponceau 5. Quantitation is then performed by scanning the protein bands on a densitometer.
Albumin Alpha-1 Alpha-2 Beta P.O.A