RESUMEN
A new mechanism of selective transport and localization of proteins inside any living cell is presented. The mechanism is based on pH-induced protein trapping. It is shown that spontaneous and unique spatial redistribution of different proteins is possible in any aqueous solution with stable non-uniform distribution of H(+) ions. This phenomenon was observed in artificial systems with fixed non-uniform pH distribution and in living cells.
Asunto(s)
Concentración de Iones de Hidrógeno , Transporte de Proteínas , Animales , Densitometría , Geles , Proteínas Fluorescentes Verdes , Cinética , Muramidasa/química , Muramidasa/metabolismo , Mioglobina/química , Mioglobina/metabolismo , Ficocianina/química , Ficocianina/metabolismo , Análisis por Matrices de ProteínasRESUMEN
A comparatively new procedure is described for the nonlinear electrophoresis of proteins. Movement and separation of complexes formed by proteins and ionic detergents is first experimentally demonstrated for SDS rainbow colored protein molecular weight markers (Amersham). This result was revealed by SDS-PAGE in an asymmetric zero average pulsed electric field with a peak amplitude of up to 300 V cm(-1) and a frequency of 100 Hz. The highest molecular weight fractions were found to have the highest nonlinear drift velocity. A two-dimensional map of distribution of the protein complexes developed using nonlinear electrophoresis followed by SDS gel electrophoresis in an orthogonal direction, reveals nonuniform distribution of the fractions. Nonlinear electrophoresis can be run without electrode chambers, since the buffer electrolyte is not used up in alternating electric fields. Thus, this new type of electrophoresis can have advantages in microfluidic systems and biochips. Also possible uses are discussed of nonlinear electrophoresis via nonlinear focusing of protein-detergent complexes for further improvement of the SDS-PAGE technique for the separation and examination of these large hydrophobic complexes.
RESUMEN
The present paper reports the nonlinear electrophoretic focusing techniques developed after an original idea by Chacron and Slater [Phys. Rev. E 56, 3436 (1997)]. Focusing of DNA molecules is achieved in an alternating nonuniform electric field, created in a wedge gel with hyperbolic boundaries. The fractions separated on such a wedge retained their rectilinear shape during the electrophoresis. Experiments with gel electrophoresis confirm the possibility of a noticeable nonlinear focusing of DNA molecules.
Asunto(s)
ADN/química , ADN/efectos de la radiación , Campos Electromagnéticos , Electroforesis en Gel Bidimensional/métodos , Modelos Químicos , Modelos Moleculares , Dinámicas no Lineales , Bacteriófago lambda/química , Bacteriófago lambda/genética , Simulación por Computador , ADN/aislamiento & purificación , ADN Viral/química , ADN Viral/aislamiento & purificación , ADN Viral/efectos de la radiación , Electroquímica/métodos , Sensibilidad y EspecificidadRESUMEN
Effects of nonlinear dependence drift velocity of (double-stranded) DNA vs. electric field strength were investigated. In comparatively weak fields, the molecular drift velocity is proportional to the external electric field, while in strong fields there is additional nonlinear component. This effect offers possibilities to manipulate the total drift velocity at will-the macromolecules of different size can be made to move in opposite directions in pulsed field gel electrophoresis.A new approach for focusing DNA molecules based on nonlinear electrophoresis and geometric trapping in electric fields is proposed. The focusing is carried out in an alternating nonuniform electric field, created by using a wedge gel with hyperbolic boundaries. It is shown that the fractions separated in such wedge retain their rectilinear shape. Gel electrophoresis experiments supported the possibility of a pronounced nonlinear focusing of DNA molecules. This nonlinear separation technique presents encouraging prospects for micromanipulating systems and also for preparative isolation of long DNA fragments and development of new separation methods for bacterial fingerprinting.
Asunto(s)
Electroforesis en Gel Bidimensional/instrumentación , Electroforesis en Gel Bidimensional/métodos , Focalización Isoeléctrica/instrumentación , Focalización Isoeléctrica/métodos , Biofisica/métodos , ADN/química , Modelos Teóricos , Factores de TiempoRESUMEN
A new approach to focusing DNA molecules in a nonuniform electric field based on nonlinear mobility (L. L. Frumin, S. E. Peltek, S. Bukshpan, V. V. Chasovskikh and G. V. Zilberstein, PhysChemComm, 2000, 11) is proposed. The focusing is carried out in an alternating nonuniform electric field, created by using a wedge gel with hyperbolic boundaries. Methods of the theory of analytical functions were used to demonstrate that the fractions separated electrophoretically in such a wedge retain their rectilinear shape. Solutions for the focusing points for the case of mere velocity cubic nonlinearity were obtained as well as for the square velocity nonlinearity, suggested in a number of modern approaches. Gel electrophoresis experiments supported the possibility of a pronounced nonlinear focusing of DNA molecules. This nonlinear separation technique presents encouraging prospects for preparative isolation of long DNA fragments and development of new separation methods for bacterial fingerprinting.
Asunto(s)
ADN/aislamiento & purificación , Focalización Isoeléctrica/métodos , Bacteriófago lambda/genética , ADN Viral/análisisAsunto(s)
Dermatoglifia del ADN/métodos , ADN/análisis , ADN/química , Electroforesis en Gel de Campo Pulsado/métodos , Bacteriófago lambda/genética , ADN de Hongos/análisis , ADN de Hongos/química , ADN Viral/análisis , ADN Viral/química , Peso Molecular , Saccharomyces cerevisiae/genética , Factores de TiempoRESUMEN
The focusing problem under isoelectric focusing has been solved analytically precisely. The solutions determine the law of the fraction moving and narrowing in the instant electric field gradient. This is especially actually because of developing the calculation methods in electrophoretical experiment.