Size-exclusion high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis...

When size-exclusion HPLC (SE-HPLC) is applied for the separation of hydrophilic and hydrophobic proteins, numerous problems can be encountered, which may present considerable difficulties. A major source of such complications is interaction between column packing and sample, especially on the so-called "Diol" and "Polyol" columns. In many cases interaction can be reduced only by adding detergents. Calibration proteins and hydrophobic membrane proteins of liver are separated by SE-HPLC. The influence of detergents on association and dissociation of protein subunits and protein configuration is shown. These factors can affect the elution volume during chromatography. Furthermore it is shown that a direct comparison can be drawn between the protein separation by SE-HPLC on the one hand and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on the other hand. As an example the separation of the delta-subunit of the acetylcholine receptor was shown under reducing and nonreducing conditions.