Protein electrophoresis is an analytical technique used to separate and analyze proteins in complex mixtures, such as blood serum. This separation relies on the principle that charged molecules move when placed in an electric field. By applying an electric current across a supporting medium, proteins migrate at varying speeds, allowing scientists to characterize and quantify them. This method is used widely in scientific research and clinical diagnostics.
The Underlying Principles of Separation
The movement of a protein during electrophoresis is determined by a combination of physical forces acting upon it within the electric field. The primary driving force is the protein’s net electrical charge, which causes it to migrate toward the electrode of the opposite charge. Since proteins are composed of amino acids with ionizable side chains, their overall charge is highly dependent on the pH of the surrounding buffer solution.
If the buffer pH is higher than the protein’s isoelectric point, the protein will carry a net negative charge and move toward the positive electrode (anode). Conversely, if the buffer pH is lower than the protein’s isoelectric point, it will become positively charged and move toward the negative electrode (cathode). The speed of migration is directly proportional to the strength of this net charge.
A second factor influencing movement is the protein’s size and shape as it moves through the support medium, typically a gel made of polyacrylamide or agarose. This gel acts like a molecular sieve, creating frictional resistance that slows down the proteins. Larger proteins experience more drag and move slower than smaller proteins, providing separation based on mass. The final separation pattern results from the interplay between the electrical pull and the physical resistance of the gel matrix.
Common Methodologies and Variations
Different analytical goals require different electrophoretic approaches. One common technique is Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). In this method, the anionic detergent SDS denatures the proteins, unfolding them and coating them with a uniform negative charge. This ensures the proteins are separated almost exclusively by their molecular size, as the intrinsic charge is masked.
A contrasting approach is Native PAGE, which is performed without the use of denaturing agents like SDS. Since the proteins remain in their natural, three-dimensional folded state, this method separates them based on both their size and their native electrical charge. Native PAGE is useful when the biological activity or structure of the protein must be preserved for subsequent analysis.
Isoelectric Focusing (IEF) separates proteins based on their isoelectric point (pI). A stable pH gradient is established within the gel matrix, ranging from acidic to basic. As current is applied, proteins migrate until they reach the point in the gradient where their net charge is zero, stopping their movement. This allows for high-resolution separation based on subtle differences in the protein’s amino acid composition.
How Electrophoresis is Used in Healthcare
In clinical settings, protein electrophoresis is most frequently applied to blood serum in a test called Serum Protein Electrophoresis (SPEP). The test separates the total protein into five distinct fractions: albumin, alpha-1 globulin, alpha-2 globulin, beta globulin, and gamma globulin. Albumin, the most abundant protein in serum, typically shows the largest and fastest-moving peak on the resulting graph, known as an electropherogram.
Changes in the relative amounts of these fractions can indicate various disease states. For instance, an increase in the gamma globulin fraction often suggests chronic inflammation or an autoimmune disorder. Abnormal spikes in the gamma or beta region, known as monoclonal spikes or M-proteins, are highly characteristic of plasma cell disorders like multiple myeloma.
Conversely, a decrease in albumin and certain globulins can point toward conditions such as liver disease, where protein synthesis is impaired, or kidney disease, where proteins are lost in the urine. Analyzing the pattern of these five fractions provides healthcare professionals with specific insights into a patient’s immune system function and overall health. The SPEP test is a valuable diagnostic and monitoring tool, helping to track the progression of disorders.