Purification and separation of hydrophobic serum amyloid A precursor isoforms by a one-step preparative method.
The levels of two major serum amyloid A precursor isoforms, SAA1 and SAA2, which are associated with high-density lipoproteins (HDL) are increased during inflammation. The hydrophobic character and the small size difference--corresponding to just 0.8 kDa--between these two members of the SAA family hinder their separation and purification on a large scale by conventional methods. In the current work, both mouse SAA proteins were purified from HDL-SAA and acute-phase serum within 10 h in a one-step procedure using the high-resolution, continuous-elution preparative gel electrophoresis Prep-Cell system in combination with Tris/Glycine SDS-PAGE. Moreover, applying the Tris/Tricine system on the Prep-Cell resulted not only in purification of the SAA proteins, but also in their separation within 16 h. The SAA isoforms were freed from SDS using a Centricon concentrator and were identified using monoclonal antibodies. Optical density profile plots of gel protein or Western blot bands in combination with a colorimetric spectrophotometric protein assay showed that the recovery of the isoforms ranged from 38% to 60%. These results show that the preparative gel electrophoresis system Prep-Cell is a suitable device for separating SAA1 and SAA2 proteins in a simplified, convenient, and fast procedure, which can be applied on a small or large scale.
Ham
Daniela
D
Institute of Preventive Medicine, Jostova 10, 66 243 Brno, Czech Republic. hamdaniela@yahoo.com
Karska-Wysocki
Barbara
B
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D057927:Hydrophobic and Hydrophilic Interactions; D051379:Mice; D020033:Protein Isoforms / Q000302:isolation & purification*; D011498:Protein Precursors / Q000302:isolation & purification*; D000685:Serum Amyloid A Protein / Q000302:isolation & purification*; D016622:Silver Staining / Q000379:methods
D020033:Protein Isoforms; D011498:Protein Precursors; C489995:Saa2 protein, mouse; D000685:Serum Amyloid A Protein