Biochemical methods of separation and characterization of proteins
Crossed immunoelectrophoresis of 2 microlitres of normal human serum. The electrophoresis was performed in thin layers of agarose; the pictured gel is about 7x7 cm. The lower part is the first dimension gel without antibodies, where the serum was applied into the slot at the lower left. The upper part is the second dimension gel with Dako antibodies against human serum proteins. More than 50 major serum proteins can be named.Fused rocket immunoelectrophoresis of an affinity chromatographic separation of human serum proteins on con A. A 15 microlitre sample from each fraction was applied (starting from left) and allowed to diffuse about one hour, then electrophoresis was performed overnight. Peak "a" was not retained, while peak "b" was retained and eluted with methyl-mannose. The arrow indicates some slightly retained proteins.
Immunoelectrophoresis is a general name for a number of biochemical methods for separation and characterization of proteins based on electrophoresis and reaction with antibodies. All variants of immunoelectrophoresis require immunoglobulins, also known as antibodies, reacting with the proteins to be separated or characterized. The methods were developed and used extensively during the second half of the 20th century. In somewhat chronological order: Immunoelectrophoretic analysis (one-dimensional immunoelectrophoresis ad modum Grabar), crossed immunoelectrophoresis (two-dimensional quantitative immunoelectrophoresis ad modum Clarke and Freeman or ad modum Laurell), rocket-immunoelectrophoresis (one-dimensional quantitative immunoelectrophoresis ad modum Laurell), fused rocket immunoelectrophoresis ad modum Svendsen and Harboe, affinity immunoelectrophoresisad modum Bøg-Hansen.
