Native electrophoresis to monitor chemical modifications of human interleukin-3.
Native electrophoresis on the Pharmacia PhastSystem using a new buffer system is used to monitor chemical modifications of human interleukin-3. The method is faster, more gentle and more convenient than amino acid analysis. Micrograms of protein suffice in contrast with milligrams required in spectrophotometric methods. As compared to generally used native electrophoresis, the combination of a changed conductivity, lower pH and the low density matrix results in a much better separation and detection of both the reaction products containing a high charge and the reaction products containing a low charge. This enables monitoring of extensive modifications as well as mild modifications. Our method also provides indications for denaturation of the protein. It has proven to be highly reproducible for at least 10 different modification reactions. Information can be obtained about the presence of the various distinct forms of the modified molecule, for instance leading to the average number of groups on the molecule. It also detects modifications not detectable with generally used native electrophoresis.
TNO Medical Biological Laboratory, Rijswijk, The Netherlands.
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D002942:Circular Dichroism; D004586:Electrophoresis / Q000379:methods*; D006531:HEPES; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007377:Interleukin-3 / Q000737:chemistry*; D014325:Tromethamine / Q000031:analogs & derivatives
D007377:Interleukin-3; D014325:Tromethamine; C026272:Bistris; D006531:HEPES