ABSTRACT
Immobilized metal affinity electrophoresis (IMAEP) is a straightforward method in which metal ions are embedded in a polyacrylamide gel strip with a negligible electrophoretic migration. Due to the preferential binding between metal ions and the phosphate group, this method uses immobilized metal ions like iron, manganese, aluminum, or titanium to capture phosphoproteins from a mixture of phosphoprotein and nonphosphoproteins. IMAEP has also been incorporated into a traditional two-dimensional (2D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) system (isoelectric focusing-PAGE) to increase its resolving power. In 2D IMAEP, the metal ions in polyacrylamide gel strip are overlaid on top of the second dimensional polyacrylamide gel to stop electrophoretic migration of phosphoproteins. Data shows that there is no detrimental effect of SDS in IMAEP on the extraction of phosphoproteins from a mixture of proteins. In addition, SDS exposes phosphate groups by unfolding the phosphoproteins to facilitate metal ion-phosphate binding while supplying the protein with negative charges.
Subject(s)
Caseins/isolation & purification , Coordination Complexes/chemistry , Buffers , Carbonic Anhydrases/chemistry , Carbonic Anhydrases/isolation & purification , Caseins/chemistry , Cell Line, Tumor , Coloring Agents/chemistry , Electrophoresis, Gel, Two-Dimensional/methods , Hemoglobins/chemistry , Hemoglobins/isolation & purification , Humans , Isoelectric Point , Metals/chemistry , Molecular Weight , Protein Binding , Rosaniline Dyes/chemistry , Serum Albumin/chemistry , Serum Albumin/isolation & purification , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
A two-dimensional immobilized metal affinity electrophoresis method is described here. In this method, ferric ions are immobilized in the second-dimensional polyacrylamide gel to extract the phosphoprotein ß-casein from a mixture containing proteins with a broad range of pI and MW. Native 7.5-15% gradient tris-glycine gel with SDS tris-glycine gel running buffer are used so that proteins can be separated according to their molecular mass in the second dimension.
Subject(s)
Electrophoresis, Gel, Two-Dimensional/methods , Electrophoresis, Polyacrylamide Gel/methods , Phosphoproteins/isolation & purification , Caseins/isolation & purification , Caseins/metabolismABSTRACT
An immobilized metal affinity electrophoresis (IMAEP) method is described here. In this method, metal ions are immobilized in a native polyacrylamide gel to capture phosphoproteins. The capture of phosphoproteins by IMAEP is demonstrated with immobilized metals like iron, aluminum, manganese, or titanium. In the case studies, phosphoproteins alpha-casein, beta-casein, and phosvitin are successfully extracted from a protein mixture by IMAEP.
