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Micropreparative isoelectric focusing protein separation in a suspended drop
Author(s) -
EgatzGomez Ana,
Thormann Wolfgang
Publication year - 2011
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201000684
Subject(s) - myoglobin , chemistry , chromatography , isoelectric focusing , isoelectric point , electrophoresis , drop (telecommunication) , analytical chemistry (journal) , cytochrome c , electrode , electrolysis , cathode , anode , biochemistry , telecommunications , computer science , electrolyte , enzyme , mitochondrion
IEF protein binary separations were performed in a 12‐μL drop suspended between two palladium electrodes, using pH gradients created by electrolysis of simple buffers at low voltages (1.5–5 V). The dynamics of pH gradient formation and protein separation were investigated by computer simulation and experimentally via digital video microscope imaging in the presence and absence of pH indicator solution. Albumin, ferritin, myoglobin, and cytochrome c were used as model proteins. A drop containing 2.4 μg of each protein was applied, electrophoresed, and allowed to evaporate until it splits to produce two fractions that were recovered by rinsing the electrodes with a few microliters of buffer. Analysis by gel electrophoresis revealed that anode and cathode fractions were depleted from high p I and low p I proteins, respectively, whereas proteins with intermediate p I values were recovered in both fractions. Comparable data were obtained with diluted bovine serum that was fortified with myoglobin and cytochrome c.