Patent Application
20060160127
The present invention relates to extraction of molecules from a separation medium.
A large variety of different separation techniques for molecules like nucleic acids, polypeptides or small non-polymeric organic molecules is known. All of these separation techniques, for example isoelectric focusing, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), native gel electrophoresis or paper electrophoresis require the recovery of the separated molecules from the respective separation media after the separation was carried out. The recovery normally involves several different steps like detecting the molecules or molecule bands in the separation media, for example by different staining techniques, followed by manually or automated excising of the molecule bands and subsequent applying of solvent to the excised molecule bands in order to recover the molecules in a liquid phase. The molecules in the liquid phase might be further subjected to one-dimensional or multi-dimensional liquid chromatography or other separation techniques for further separation. Analysis of the separated molecules can be carried out using mass spectroscopy.
It is an object of the present invention to provide an improved extraction technique. This is solved by the independent claims. Favorable embodiments are subjects of further claims. Thus, embodiments of the invention provide a fast and easy to use extraction technique, which can enable to extract different molecule bands from a separation medium in one step.
According to an embodiment, extraction of molecules from at least one separation medium comprises:
Preferred embodiments provide a fast and easy-to-handle procedure for the extraction of large amounts of different molecules, located in different areas of the at least one separation medium in one step. Due to the (preferably large) number of compartments of the frame, which divide the separation medium into different compartments with different molecules, the different molecule bands in the separation medium do not have to be processed separately anymore but can be extracted in one step by applying solvents into the different compartments. Furthermore, the different molecule bands do not have to be excised from their respective separation media anymore.
Normally, the molecules are initially separated in the separation medium and are then subjected to A). As mentioned above, different state-of-the-art separation techniques like isoelectric focusing, sodium dodecyl sulphate polyacrylamide gel electrophoresis or paper electrophoresis can be used.
In another variant, a bottomless micro-titer plate with separate compartments is used. Micro-titer plates are easy to produce and are current state-of-the-art instruments, therefore providing a cheap instrument for the separation of the separation medium into different compartments.
In a preferred embodiment, prior to A) the molecules are separated in a plurality of separation media located on a carrier. Afterwards, in A) the frame is brought in contact with the plurality of separation media, separating the plurality of separation media into different compartments.
This provides the possibility to process many separation media loaded with a large amount of molecules in one step after separation of the molecules by extracting the separated molecules in the different separation media in one step by using the frame with the different compartments. Therefore, this enables an easy extraction of molecule bands located in different separation media in one step.
It is also possible to use gel-based separation media. The gel-based separation media might be selected from polyacrylamide, agarose, dextran or starch. These separation media are useful for the separation of polypeptides, nucleic acids or other small organic molecules. Paper-based separation media can also be used, for example when a paper electrophoretic separation of molecules is carried out.
In a preferred embodiment, the at least one separation medium is furthermore subjected to motion in B). Motion during the extraction of the molecules in B) can support the extraction process by e.g. equally distributing the already extracted molecules in the liquid phase so that no local high concentrations of the molecules can occur. Therefore, motion like shaking or other means keeping the extraction medium in motion e.g. stirring or rotation might enable a continuous diffusion of the molecules from the separation medium into the solvent. It is also possible that the motion might disrupt the integrity of the separation medium, therefore simplifying the extraction of the molecules.
In another variant, in B) a voltage is applied to the at least one separation medium. The voltage also might ensure a good extraction process.
Advantageously, in A) a positioner for sealing and positioning the frame onto the at least one separation medium is applied. The positioner can e.g. comprise a force, which can be applied, pressing the frame onto the separation medium ensuring a stable positioning of the frame on the separation medium with no relative movement of the frame to the separation medium during the extraction procedure. Therefore, no slipping can occur, which might lead to mixing of molecules from different compartments.
An apparatus for separation and extraction of molecules from at least one separation medium comprises:
at least one separation medium and
a frame with compartments for separation of the separation medium into different compartments.
Such apparatus is useful for carrying out a separation of molecules e.g. polypeptides, nucleic acids or other small organic molecules using a large variety of different separation techniques like polyacrylamide gel electrophoresis, agarose gel electrophoresis or other electrophoretic separation techniques. After the separation, the molecules are normally located in different areas of the separation medium, which preferably has a strip-like form. The frame with the different compartments can then be used for separation of the different molecules in different areas of the separation medium by bringing the frame in contact with the separation medium, thereby dividing the separation medium into different compartments.
This apparatus can be especially useful for separation and extraction of large amounts of polypeptides by e.g. isoelectric focusing or polyacrylamide gel electrophoresis. The apparatus can also be used for separation of nucleic acids in an e.g. agarose gel-based separation medium. Once the position of the molecules of interest in the separation medium after the separation procedure is known, different kinds of molecules can be isolated from each other and extracted from the separation medium in a very simple and fast way.
Preferably, the apparatus comprises a plurality of strip-like separation media located on a carrier for simultaneous separation of molecules in one step. Such apparatus enables a fast and easy-to-handle procedure for separation of large amounts of molecules at the same time in a single step. Due to the frame with the different compartments, such an apparatus also allows a fast extraction procedure of the separated molecules from the plurality of strip-like separation media. The plurality of separation media can, for example, comprise polyacrylamide-based gel strips with immobilized ampholytes for isoelectric focusing procedures of polypeptides, agarose gel strips for separation of nucleic acids or polyacrylamide-based separation media for the separation of polypeptides.
Advantageously, the frame furthermore comprises a seal for sealing the different compartments. The seal can e.g. comprise flexible sealing bands e.g. rubber bands, which are present in the areas of the frame contacting the separation medium (see, for example,
The apparatus can furthermore comprise a positioner for positioning the frame on the separation medium. The positioner can, for example, comprise a clamp, which fixes the frame on the separation medium and prevents a slipping of the frame relative to the separation medium during the extraction procedure (see for example,
In the following, embodiments will be explained in more detail with reference to the figures. All figures are just simplified schematic representations presented for illustration purposes only.
The FIGS. 2 to 4 show cross-sectional views of a frame with different compartments located on different separation media.
Lyophilized Escherichia coli cells (strain B-ATCC 11303, Sigma) were suspended in buffer (7M urea, 2M thiourea, 4% CHAPS, 1% DTT) and disrupted in a BEAD-BEATER (BIOSPEC PRODUCTS) according to the recommendations of the supplier. Insoluble material was removed by centrifugation. The protein concentration of the cell extract was determined by the Bradford method and adjusted to 1 mg/ml with buffer. 0.25 ml extract were loaded on a 13 cm long Immobiline dry strip pH 4-7 (Amersham) by overnight rehydration at room temperature. Isoelectric focusing (IEF) was done using an IPGphor (Amersham) at 20° C. with a current limit of 50 μA/strip in cup loading strip holders (Amersham).
After one hour focusing at 500 V and one hour focusing at 1000 V, the voltage was set to 8000 V until a total of 20 kVh was reached. Subsequently, a frame with fifteen compartments each with a size of 0.6×0.6 mm was placed on top of the strip and 15 μl buffer was added to each compartment. The in liquid recovery was done for at least one hour with voltage (8000 V) at 20° C. or without voltage and with shaking at room temperature. The protein concentration of every fraction was measured with the Bradford method (see
The scope of the invention is not limited to the embodiments of the figures. Indeed, variations especially concerning the usage of different separation media are possible.
Embodiments of the invention can be embodied in each novel characteristic and each combination of characteristics, which includes every combination of any features, which are stated in the claims, even if this combination of features is not explicitly stated in the claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/EP03/50652 | Sep 2003 | WO | international |
