The invention relates to a system for two-dimensional gel electrophoresis, wherein in at least one dimension of the electrophoresis, the functional core part thereof is formed essentially of Corian material. Furthermore, at least one electrode is preferably designed in a mobile manner in the first dimension, so that it can be adapted to varying lengths of gel.
Prior art electrophoresis has been employed as an efficient and gentle method e.g. in the separation of proteins or amino acids, but also of whole cells. It utilizes the variable deflection of charged particles in an electric field to achieve a separation effect. The degree of deflection is primarily influenced by the strength of the field between two electrodes and by the electric mobility of the particles to be separated. The level of mobility depends on various quantities, such as charge and size of the particles to be separated, as well as pH value, viscosity and ionic strength of the buffer solution. Traditional one-dimensional methods of polyacrylamide gel electrophoresis (PAGE) are capable of separating a maximum of about 100 proteins in a single sample, for which reason they are not suitable for the analysis of complex protein mixtures, such as encountered in cell extracts.
At present, two-dimensional electrophoresis is the most efficient technique for separating complex protein mixtures. It separates the proteins according to two different parameters, i.e. the isoelectric points (pI in the first dimension) and the electrophoretic mobility (in the second dimension). There are a variety of uses of 2D electrophoresis. Thus, for example, protein patterns are analyzed in various organisms and cell types. Purities of protein fractions are examined, and isoelectric points, molecular weights of subunits and the number of isoforms are determined. The two-dimensional gel electrophoresis system, which furnishes best resolution for the separation of complex protein mixtures, combines the technique of isoelectric focusing (IEF) in the presence of urea and a neutral detergent in the first dimension with plate gel electrophoresis under denaturing conditions using sodium dodecylsulfate (SDS) in the second dimension (O'Farrell, P. H. (1975), J. Biol. Chem. 250, 4007-4021). The separating system uses two independent protein properties: one of them is the charge reflected by the isoelectric point (pI), the other one is the molecular weight which determines the mobility of the SDS-protein complexes in polyacrylamide gels. O'Farrell has demonstrated the great potential of this technique for the first time, being able to resolve more than 1,000 polypeptides in cellular extracts. However, in an IEF/SDS system using carrier ampholytes, basic proteins are not separated adequately because migration thereof into the IEF gel is generally poor. Even when using more basic ampholytes, the expanse of the pH gradient in the basic range is small because, due to the cathode drift, the actual gradient does not significantly rise above pH 7.5. Furthermore, the small number of basic proteins entering the IEF gel under these conditions invariably give rise to streaks. However, this problem can be avoided by using the non-equilibrium pH gradient electrophoresis (NEPHGE) in the first dimension, as described by O'Farrell.
The main difference between NEPHGE and IEF is that the samples in the former case are applied to the acid side of the gel and that the voltage×time product is smaller than that in the IEF. Under such conditions, the pH gradient does not reach full equilibrium. As a result, the proteins are not completely focused at their isoelectric point, as is the case in the IEF. Nevertheless, most proteins are separated according to their differing charges in NEPHGE gels as well.
The well-known systems used in two-dimensional electrophoresis suffer from a number of drawbacks: regarding safety, the first dimension is not sufficiently protected from the high voltages being used; furthermore, many systems are required for the different lengths of gel, and, in addition, the apparatus for the second dimension are not easy to handle; for example, they are not stackable, thus highly space-demanding, and consume a lot of chemicals.
The invention solves this problem by means of a system in accordance with the claims.
In the inventive system for two-dimensional gel electrophoresis, at least one functional core part is formed essentially of Corian material. For example, the functional core part can be the plate on which the gel is positioned in the second dimension. The Corian material may have drill-holes incorporated therein which represent the cooling system. In this way, the system can adopt a more compact design. More specifically, the apparatus for the second dimension can be stacked horizontally and consume less chemicals.
The system comprises at least two components for the first and the second dimension of the electrophoresis, respectively, the first dimension comprising an electrode which can be adapted to different lengths of the gels, the first dimension being surrounded by a jacket body made of plexiglass, comprising at least one door with an NO contact for a circuit, and the second dimension of the gel electrophoresis being made essentially of Corian material. Such a system is advantageous in that the circuit is interrupted when opening the door or doors and closed when locking the doors. For example, interruption of the circuit via a contact on the door and a corresponding contact at the base of the apparatus can be envisaged for the first dimension.
It is preferred that the material is Corian Glacier White.
In a preferred embodiment of the invention, at least one electrode in the first dimension is positioned on a movable carriage on a contact arm.
The invention also relates to the use of Corian for the production of a system of gel electrophoresis, especially for the production of functional components.
This material has the advantage of being malleable, formable, and thus shapeable, e.g. for integrating cavities and canals which—likewise by way of example, but not exclusively—can be used for cooling purposes. Comparable systems normally use glass which is known to be immalleable.
The above-mentioned two components used to perform the electrophoresis in the first and the second dimension are separate electrophoresis units. Therefore, according to the invention, an electrophoresis unit used to perform the first dimension of a two-dimensional gel electrophoresis is also a subject matter of the invention, comprising an electrode which can be adapted to different lengths of the gels, the first dimension being surrounded by a jacket body made of plexiglass, comprising at least one door with an NO contact for a circuit.
The invention is also directed to an additional electrophoresis unit used to perform the second dimension of a two-dimensional gel electrophoresis, which is formed essentially of Corian material. Owing to the flat design with optionally integrated cooling, it can be stacked horizontally, thus saving space to a large extent, which is particularly advantageous in situations involving narrow laboratories. Also, the consumption of chemicals is reduced to a large extent.
Without intending to be limiting, the invention will be explained below with reference to an example.
The figures show:
Advantageously, further separation of the biomolecules is effected in the second dimension as explained above.
Number | Date | Country | Kind |
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102005041638.1 | Aug 2005 | DE | national |
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/DE06/01456 | 8/18/2006 | WO | 00 | 7/10/2008 |