Biochemistry lab. A presentation was made about the serum protein electrophoresis studied. Electrophoresis is a technique in which molecules are distinguished based on their charge, molecular weight and size in an electrical field. SPE on cellulose acetate membrane From the early 1950s, classical 5-band cellulose acetate electrophoresis became popular as a standard procedure and was used for many years. It has a very good resolution when applied correctly. Agarose Gel Electrophoresis (AGE) was first put into routine use by Laurell in 1962. The separation is based on the charge/mass ratio of the protein. Agarose is obtained from a type of algae. The gel is prepared by dissolving agarose placed in electrophoresis buffer at high temperature. The boiled agarose solution is cooled to 50 degrees and poured into gel trays. The sample to be separated is loaded into wells created with combs and separated by moving in an electrical field. After the gel is properly prepared or the samples are loaded and electrically separated, they are stained and the samples are visualized. (Dye: Coomassie brilliant blue, Amido Black 10B)
Densitometric scanners are used to detect relative percentages of each protein fraction. These percentages are then multiplied by the total protein in the sample (measured separately) to obtain the protein concentration in each fraction. Solutions Used: electrophoretic buffer, sample dilution, fixation, staining. Capillary electrophoresis (CZE) Capillary electrophoresis began to be used in the mid-90s. Differences Between AGE and CZE:
• Capillary Electrophoresis: medium Water-based, fast results, good resolution, small sample amount, no staining, relatively difficult to interpret, detection limit is higher in monoclonal proteins
• Agarose Gel Electrophoresis: medium Gel, slow results, gold standard, large sample amount, staining, easier to interpret, detection limit for monoclonal proteins is lower (12 mg/dL).
• Immunofixation electrophoresis (IFE): It is a gold standard biochemical technique used in the diagnosis, follow-up and management of monoclonal gammopathies. In IFE, immunoprecipitation occurs using Ig-specific antisera along with electrophoresis, and Igs become visible as bands.