MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) PROTEIN-ANALYSIS BY OPTIMIZED 2-DIMENSIONAL ELECTROPHORETIC METHODS

Human histocompatibility molecules HLA‐Class I and Class II (DR, DQ, DP) were analysed using three two‐dimensional protocols: nonequilibrium pH gradient electrophoresis (NEPHGE), isoelectric focusing‐acidic gradient (IEF‐AG) and isoelectric focusing‐basic gradient (IEF‐BG). The three methods differ in their carrier ampholyte combinations and electrophoretic conditions. They provide different pH gradients and therefore different electrocusing profiles. The NEPHGE protocol was edequate for separating proteins across a broad range of pI mobilities, i. e. 4.4. pH units between the acidic and the basic end. In contrast, the IEF‐AG and the IEF‐BG protocols gave a sepration power across a narrow pH range, 1.9 and 1.7 pH units respectively. Thus, whereas the NEPHGE protocol provides a tool for a global major histocompatibility complex (MHC) antigen profile analysis, the IEF‐AG and ‐BG allows one to investigate subcomponents of the individual MHC chains. For example, NEPHGE analysis of the HLA Class I heavy chain revealed a single spot. However, IEF‐BG revealed the presence of six equidistantly spaced spots spanning a short pH gradient with identical molecular weight. Similar improved resolution was seen for the HLA‐DR, DQ, and DP molecules. The IEF acidic gradient was adequate for separating the alpha chain; the IEF basic gradient gave better resolution of the beta chains. This data provides a baseline set of conditions for both analytical and preparative MHC protein studies prior to amino acid sequencing. Copyright © 1990 VCH Verlagsgesellschaft mbH