Micropreparative high resolution purification of proteins by a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and membrane blotting.
We report a simple, economical, and efficient protocol for protein purification from cells. First, proteins of cell lysates were separated by standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted to protein-blotting membrane. The blots were stained with Coomassie blue or developed by immunoblotting to visualize specific proteins. The bands corresponding to those visible by immunoblotting were excised from the dye-stained blots and subjected to isoelectric focusing. The focused gel was stained with Coomassie blue. Finally, the stained bands were excised and subjected to another SDS-PAGE separation and electrotransferred back to protein-blotting membrane. At this stage, the purified proteins were suitable for microsequencing. We have tested the feasibility of this novel technique by purifying proteins with molecular weights ranging from 19 to 100 kDa from a lysate of Sarcocystis neurona, the etiologic agent of equine protozoal myeloencephalitis. The purity of proteins was demonstrated by reverse-phase high-performance liquid chromatography. Partial sequences of these purified proteins were obtained by N-terminal or digestive sequencing.
Liang
F T
FT
Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington 40546, USA.
Granstrom
D E
DE
Timoney
J F
JF
Shi
Y F
YF
D000818:Animals; D000953:Antigens, Protozoan / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D007525:Isoelectric Focusing; D008567:Membranes, Artificial; D011506:Proteins / Q000302:isolation & purification*; D012522:Sarcocystis / Q000276:immunology
D000953:Antigens, Protozoan; D008567:Membranes, Artificial; D011506:Proteins