Optimization of serum electrophoresis by utilization of the Blue-native technique

Abstract

The method of choice for the electrophoretic separation of proteins is, to this day, SDS-PAGE. Proteins and protein complexes are separated under reducing conditions, i.e. in their denatured form. In comparison, the method of Blue-native PAGE allows the separation of native proteins. Even though the method has already been introduced in the early 1990s, little research on application and optimization of Blue-native PAGE has been done since that time. The molecular weight resolution of SDS-PAGE is very limited as it is complicated to resolve molecules larger than 250 kDa. This thesis deals with a methodological modification in order to overcome this disadvantage, by optimizing the electrophoretic setup. An optimal gradient for covering a molecular weight range up to 1200 kDa was developed and reproducibility of the method was evaluated. In order to prove the new method, protein complexes as occurring (only) in human blood were separated: In a first dimension in their intact (native) form by Blue-native PAGE, followed by a reducing SDS-PAGE as the second dimension. Hereby samples of patients suffering from rheumatoid arthritis, a disorder where immune complexes are formed excessively on an autoimmune base, were investigated as described. Results, i.e. grouping according to electrophoretic pattern recognition, showed a clinically significant coincidence to grouping according to clinical parameters plus an additional subgrouping option based upon the separation of IgG complexes.