PatentDe  


Dokumentenidentifikation EP1801573 09.08.2007
EP-Veröffentlichungsnummer 0001801573
Titel Vorrichtung und Verfahren für Parallele Zweidimensional-Elektrophorese
Anmelder F. Hoffmann-La Roche AG, Basel, CH;
Roche Diagnostics GmbH, 68305 Mannheim, DE
Erfinder Curcio, Mario, 6048 Horw, CH
Vertreter derzeit kein Vertreter bestellt
Vertragsstaaten AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HU, IE, IS, IT, LI, LT, LU, LV, MC, NL, PL, PT, RO, SE, SI, SK, TR
Sprache des Dokument EN
EP-Anmeldetag 21.12.2005
EP-Aktenzeichen 050280338
EP-Offenlegungsdatum 27.06.2007
Veröffentlichungstag im Patentblatt 09.08.2007
IPC-Hauptklasse G01N 27/447(2006.01)A, F, I, 20070529, B, H, EP
IPC-Nebenklasse B01L 3/00(2006.01)A, L, I, 20070529, B, H, EP   

Beschreibung[en]

The present invention refers to an arrangement for the separation of a sample mixture for analytical reason, based on two-dimensional gel electrophoresis according to the introduction of claim 1 and a method for gel electrophoresis analysis suggesting ways for integration and automation.

In particular the present invention relates to advantageous embodiments for gel electrophoresis, providing convenient and effective means of achieving parallel analysis and comparative studies, by extending the scope of the method at the basis, already disclosed in the non published patent application EP 05007912.8 attached as appendix A, based on fast UV gel polymerization and SDS electrokinetic equilibration without the use of valves.

Background

Two-dimensional slab gel electrophoresis is still the most used approach to proteomics and it might be still for several years, if other limitations still present are addressed. Indeed, this remains a time-consuming and laborious procedure, requiring trained personnel, on the hands of whom the quality of results is mainly depending. Although the post-electrophoretic steps are highly robotized, the separation step is far from it, so that problems with accuracy and consistency can arise from variations in the numerous parameters to keep under control. Some of these are for example, sample loading and rehydration, in terms of sample amount, losses, and homogeneity of the strip, strip handling with risk of damaging and contamination, imprecise and slow coupling of the strip to the gel, gel casting and polymerization, in terms of homogeneity, casting and reaction speed, especially for gradients, air sensitivity, time for completion until run is started, risk to trap bubbles causing consequently also field discontinuities, increase in temperature during the run, pH and viscosity changes, loss of buffer capacity. Lack of acceptable reproducibility, meaning that no two gel images are directly superimposable, remains therefore a major problem if considered that gels are mostly made to be compared, e.g. to detect and quantify differences in protein expression between experimental pairs of samples. In practical terms, this translates in the need to run more gels to build reference maps for each condition and reach a certain degree of certainty, which in turn means even more manual work.

A technique, apparently overcoming this problem, was introduced in 1997, namely fluorescent 2-D differential gel electrophoresis (DIGE) [1]. This is based on the use of two mass- and charge-matched N-hydroxy succinimidyl ester derivatives of the fluorescent cyanin dyes Cy3 and Cy5, possessing distinct excitation and emission spectra, to differentially label lysine residues of two protein samples, which are then mixed and run on the same gel. Thus, matching is automatic and straightforward and in principle only a single gel could be sufficient. However, for a proper statistical evaluation, at least three to five gels are required as well. To make things even more complicated than before is the fact that very stringent labeling conditions should be followed. It is indeed well known that pre-labeling can generate a large number of positional isomers as well as partially reacted species yielding very heterogeneous results. Labeling must be therefore minimal, trying to achieve possibly the addition to a single lysine residue on the entire protein molecule. In addition, the over-reacted species might precipitate as a result of an acquired increased hydrophobicity, but the biggest issue is the fact that one cannot simply run a DIGE gel and cut out the spots of the differentially expressed proteins for subsequent MS analysis. Indeed, there is no way to predict to which lysine, thus to which peptide of the digested spot, the covalent fluorescent label will be attached, so that peptide identification might be problematic. Moreover, after the gel has been removed from the fluorescence scanner, the spots will no longer be visible, so that Sypro Ruby or other visual staining technique should be thus used anyway for the post-electrophoretic visualization. Finally, perhaps the biggest limitation is represented by the very high cost of the equipment, software and reagents.

Automation associated with better reproducibility are the main strengths of the instrumental chromatographic approach, as no further manual intervention is required after the sample has been loaded. Nevertheless, this is true only when using the same column and running the same method sample after sample in a sequential order. Columns of the same size packed with the same material might give indeed different elution times, as column packing is per se not perfectly reproducible. New materials as e.g. monoliths bring with them new advantages but columns are still made one by one, meaning that, in analogy to gels, no two chromatograms run in parallel are superimposable. Besides this, limitations due to cost and complexity of instrumentation make this approach after all not faster and not really more convenient, despite other inherent advantages like on-line detection and the possibility of direct coupling to MS. Gels, on the other hand, can be easily run in parallel, can offer under optimal conditions superior resolution, and can be directly compared by imaging. The potential is therefore still very big if integration and automation, thus higher reproducibility and throughput, are achieved for gels too, allowing to run more and comparable gels in less time with less work and reduced costs.

In the previous not yet published application EP 05007912.8 , a modification of the general method is introduced, based on the combination between valveless first dimension, SDS ek equilibration, and fast UV polymerization, ultimately leading to a truly simple integrated and automated system and other important advantages over the prior art, the most important of which are recalled and described again below.

For simplification reasons and for the better understanding of the invention as proposed within the EP 05007912.8 the various method or process steps for the two-dimensional gel electrophoresis analysis are described in operational sequence. Below is a brief list of the steps involved during the execution of the developed method followed by a discussion for each of them:

  1. 1. Perform reduction/alkylation prior to IEF (isoelectric focusing)
  2. 2. Load sample.
  3. 3. Run IEF in any of the following proposed ways.
  4. 4. Increase spacing between strip and opposite surface of the gel mold.
  5. 5. Bring gel solution for 2nd dimension separation while achieving coupling at the same time and polymerize.
  6. 6. Bring SDS to the focused proteins electrokinetically.
  7. 7. Replace running buffer and run 2nd dimension.
  8. 8. Open gel mold to remove gel.
  9. 9. Proceed with fixing and staining.

Within the following description of the various steps also reference is made to the attached drawings 1 - 7, in which examples of possible embodiments and parts of the developed system or device respectively according the EP 05007912.8 are shown.

Step 1

Reduction/alkylation is performed just before sample loading as the last step of the sample preparation according to [2]. Same reducing and alkylating reagents, i.e. tributylphosphine (TBP) and vinyl pirydine (VP) are preferably used, although with a slight modification of the method. It has been found that it is not necessary to buffer the sample solution for the alkylation reaction to occur, thus avoiding a useless increase of the salt concentration that would result in high current and longer IEF times unless desalting is carried out. Moreover, it is considered more efficient to add TPB and VP in two consecutive steps rather than simultaneously since the two reagents can react with each other. In this way, shorter reaction times, e.g. overall 30 min, are also needed. As an example, a typical solution used to solubilize the protein sample, with variations of course allowed, consists of: Thiourea 2 M Urea 7 M CHAPS 2 % (w/v) Bio-Lyte® 3/10 Ampholytes 0.5 % (v/v) Bromophenol Blue 0.002 % (w/v) 1,2 - propandiol 20 %

To this, TBP is added e.g. first in concentration of 5 mM for about 10 min, followed by addition of VP 20 mM final concentration for about 20 min and again TBP in sufficient molar amount to quench the excess of the previous reagent, rather than a different reducing agent such as dithioerythryol (DTE).

The function of the 1,2 - propandiol, which is a favorite additive among others possible as e.g. glycerol, PEG, diethylenglycole, is to minimize EOF during IEF while maintaining the viscosity of the sample solution low, which is important for the sample loading step as will be seen below.

Step 2

The sample, e.g. in the solution above, is inserted such as e.g. pipetted into a small sample well from which the sample can get in contact with the strip and the internal surface of the disposable body directly facing the strip and be guided as proposed according to the present invention by capillary hydrophilic forces between such surface and the semi-dry strip filling entirely the volume so defined and shown in Fig. 1. Fig. 1 shows in longitudinal section part of the first gel strip as proposed in the EP 05007912.8 arranged within a 2D gel electrophoresis device or disposable respectively. The sample 1 as described above is inserted in a sample well 3 and is guided along a capillary opening 5 along the hydrophilic gel strip 7 in the direction of the shown arrow. Preferably, but not necessarily the area in correspondence of the strip is hydrophilic, while at least part of the rest of the surface 9 of the disposable body 11 is hydrophobic or otherwise non gel sticking. Contribution to sample guiding might be given simply also by two drawn parallel lines on the disposable body reproducing the size of the strip underneath. Gel sticking might be desirable on the same cover plane where the strip is attached, which can then be all hydrophilic or have gel bond properties. If this is a e.g. foil, the advantage is that at the end it can be peeled together with the gel, making handling easier and minimizing the risk of breakage. Pressure or vacuum may be employed to assist the loading but can in general be avoided. In this controlled way, a volume of sample corresponding exactly to the amount needed to rehydrate the strip can be introduced minimizing waste.

Step 3

To be noticed is the fact that the strip 7 has not to be closed at its sides by any valves. Evaporation is minimized because the gel mold is nearly closed at all sides and because temperature is preferably kept cool during IEF being the disposable positioned e.g. on a cooling plate. Commercially available strips can be used, which would be already integrated in the closed compact disposable or otherwise separately supplied attached to the cover, which would close the main disposable body. Strips may also be polymerized in situ using the same system of hydrophilic guiding, this time on both surfaces, or otherwise a hydrophilic neutral porous material, e.g. a membrane with a strip shape. In this case, however, instead of passive rehydration we would have an active sample loading. Disclosed is also a new IEF medium, which might be premixed with the sample solution, guided as above to assume a strip shape and capable of gelling when increasing the temperature slightly above room temperature. A medium with this characteristic is a block copolymer of ethylene oxide and propylene oxide belonging to the class of commercially available products known as Pluronics* from BASF. A possibly suitable one is e.g. Pluronic F127 at a concentration of about 20% or above when mixed with a sample solution such as that described above. This product besides other commercial applications has already been used as efficient sieving medium in capillary electrophoresis of oligonucleotides and sometimes of peptides but was never used for IEF of proteins. A normal characteristic of this copolymer when dissolved in water solution at a critical concentration is to be liquid at low temperature, typically < 5 °C and become a sort of liquid crystalline gel at room temperature. The presence of urea, thiourea, ampholytes and detergents in the sample solution shifts the gelling point above 30-35 °C, thus making the liquid, although viscous, easy to handle and guide at room temperature. Both in capillary electrophoresis and in the shape of a strip it was possible to obtain nicely focused proteins as shown in Figures 2 and 3. Figure 2 shows a pluronic strip 15 with the separately located protein components 17 after the isoelectric focusing step. Fig. 3 shows in diagram form the separation of the same sample by capillary IEF in a pluronic filled capillary. Here, the line C represents the current drop during IEF while the line P shows the IEF peaks following mobilization. The advantage in capillary electrophoresis is that uncoated capillaries can be used due to the dynamic coating properties of the polymer itself.

Step 4

A problem experienced, at least with commercial strips, is represented by an irreproducible second dimension when the strip and the second dimension gel, polymerized directly in contact with the strip, have the same thickness. On one hand a spacing of the mold corresponding to the thickness of the rehydrated strip is necessary in order to introduce the right amount of sample, rely on a good capillary force and perform a good first dimension analysis. On the other hand a small space above the strip is required to achieve proper coupling with the gel and perform a good second dimension analysis. To solve this problem, three possible solutions are shown schematically in figs. 4 to 6, where by way of a cross-sectional view part of the analytical disposable is shown in the area of the first gel strip 7. One way is to have constant thickness for the gel mold and change thickness only in correspondence of the strip. For example, one can have a slit 21 in the disposable body 11 where a fitting bar 23 with a hydrophilic bottom 24 is automatically lowered and raised accordingly with two allowed positions as shown in Fig. 4a and b. Fig. 4b shows the raised fitting bar 23 to shape a gap 25 above the strip 7. But other variants are possible, where e.g. the strip to move is attached either on a rigid or elastic component. Another way is to change the spacing of the entire gel mold between two allowed positions. For this purpose an elastic compressible frame 27 - "O"-ring-like - can be inserted between two mold planes 12 and 14, as shown in fig. 5a and 5b and for these different geometries could be drawn. Eventually the two planes 12 and 14 can be brought to touch each other when the frame is squeezed as shown in fig. 5a, while a cavity or a gap 25 is shaped between the upper mold plane 12 and the gel strip 7 when the compressible frame is expanded, as shown in fig. 5b. A suitable cavity 25 with the same height of the strip 7 can be left in correspondence of the strip such as schematically drawn in fig. 6. Again, fig. 6a shows the compressible frame squeezed, while fig. 6b shows the compressible frame in expanded condition. The mechanism of sample loading is preferably still the same but the air volume around the strip would be reduced.

Step 5

For more controllable gel casting this step is preferably carried out vertically, which means that the instrument will operate a 90 ° rotation of the disposable. The introduction of the gel solution can occur through proper tubing fitting or needle either from the bottom to the top or from the top to the bottom and the strip may find itself located at any of the four sides relative to the vertical mold. In this way the gel solution will fill completely the mold, at least partially contacting, covering and/or enclosing the strip and it is preferable in order to maintain the resolution of the first dimension and diffusion of acrylamide inside the strip, with possible crosslinking to the sample, that polymerization occurs rapidly. For this reason the traditional method, making use of ammonium persulfate (APS) and N,N,N',N'-tetramethylethylenediamine (TEMED) as initiator and catalyst respectively of radical polymerization, is not preferred because these reagents have to be added and mixed at the last moment as they start immediately polymerization already during casting and because the reaction proceeds slowly taking normally more than one hour to be completed. Ideally, the gel solution contains already the reagents for polymerization and is stable under storage conditions; important is also that once the reaction is triggered, e.g. by external energy source, this proceeds fast, while maintaining the characteristics of the traditional sieving gel. This can be achieved for instance by UV-initiated polymerization choosing an initiator that is stable in the acrylamide gel solution until exposed to a light source whose wavelength range comprises its absorbance spectrum.

UV transparent materials should be thereby used for the disposable. As these compounds are generally not polar, hence poorly soluble in aqueous solution, a modification of the gel solution is necessary. For example up to 10 % diethylenglycole without compromising the performance of the gel can be used. A suitable initiator is for example 2, 2'-dimethoxy-2-phenyl-acetophenone (DMPA) at concentration of 0.05 % or below. By this, exposure of the gel mold to UVA light of sufficient power results in complete polymerization in less than 5 min.

Although photopolymerization itself is not new, it was never applied to our knowledge to two-dimensional gel based proteomics.

Steps 6 and 7

At this point the strip is coupled to the gel with the proteins focused in bands within the strip at their isoelectric points. This means however that carrying a zero net charge they won't be able to be transferred to the gel for the second dimension analysis. They have indeed been previously alkylated but are not yet complexed with Sodium-dodecyl-sulphate (SDS), which gives them a net negative charge and binds to them with a constant ratio allowing them to be separated now according to size through the sieving matrix of the gel. One way to bring SDS to the proteins is electrokinecally from the cathodic buffer reservoir. A concentration of SDS higher than that present in the running buffer is however necessary, e.g. 2% versus 0.1 or 0 %. This has two implications: first the buffer at the cathode needs to be replaced or diluted after electrokinetic equilibration, second the distance of the strip from the buffer should preferably be small (e.g. <5 mm) in order to minimize the zone at high SDS concentration entering the gel. The resulting effect is however superior to the standard procedure. As the SDS migrates into the gel and encounters the protein bands, these start to mobilize from the tail while the head is still steady. The result is a stacking effect with the bands gradually compacting at the opposite side of the strip before beginning their migration and separation inside the gel, which in turn means a gain in resolution. In that respect Fig. 7 shows the result of the two-D separation of an E.Coli lysate, where a total sample amount of 150 µg was loaded on an IEF strip of 7cm, pH range 4-7, and the second dimension separation was executed according to the present method based on SDS electrokinetic equilibration. The achieved resolution, shown in Fig. 7, appears clear to a person skilled in the field and was confirmed by mass spectrometry analysis, which proved also the absence of artifacts. Once proteins have complexed with SDS, the interaction is sufficiently strong so that no SDS needs actually to be present in the gel solution from the beginning. By this way we also make sure that no SDS diffuses into the strip from the gel solution causing partial complexation of the proteins and potentially disrupting the stacking effect described. SDS electrokinetic equilibration with the first buffer is preferably carried out at lower electric fields compared to the separating conditions. Applied is e.g. an electric field in the range of approximately 5 to 6 v/cm or lower. This step takes approximately 5-10 min, the time necessary for the SDS to pass through the strip, after which the run is paused e.g. for the time necessary to replace the buffer, the buffer at the cathode replaced or diluted, if starting from a smaller volume, and the run restarted at much higher electric fields for fast separation, while the heat is dissipated through efficient cooling. The strength of the higher electric field is dependent on the system and is preferably higher than e.g. approximately 20 volt per centimetre. If a higher electric field is applied, a higher cooling capacity of the system has to be applied. Preferably, the gel mold is closed from all sides between the two planes, e.g. by means of a squeezable frame as mentioned above in respect to Figures 5 and 6. The buffers contact the gel at two opposite edges of the mold and on the same plane, through two parallel slits, one of which positioned between the strip and one edge, and as close as possible to the strip for the reasons above. The slits are also preferably closed to prevent more efficiently evaporation and drying of the strip and to avoid gel solution leaking during casting in the vertical position. The slits might be created for example only when and where needed by cutting, with a blade function integrated in the instrument, thinner linings, that represent physical integral parts of the disposable body, e.g. made by injection molding. The slits could be otherwise sealed by a porous membrane, e.g. polyethylene, PES (polyethersulfone), polypropylene, or PET, with the right thickness and porosity and which withstand the extrusion pressure of the gel during casting but are then wetted by the buffer containing SDS thus establishing electrical contact with the gel. The use of tapes or adhesive tabs is preferably avoided from an automation point of view.

The way of bringing SDS electrokinetically to mobilize focused proteins is not new. There is one previous published work [4] in which however a different system is described with proteins focused first in a microfluidic channel and where SDS elctrokinetically introduced is necessary to inject separate zones into side channels. Here instead the first application to two-dimensional gel electrophoresis is reported and for the first time this stacking effect between strip and gel is described.

Steps 8 and 9

From sample loading to this point all steps could be automated. Once the second dimension run is completed, the user can remove manually the disposable from the instrument and take the gel off. Preferably, for easier handling, the gel remains attached to one of the surfaces of the mold, either the disposable body or the covering plane, which can consist either of a rigid plate, e.g. glass or polymer, or a polymeric more flexible foil. The surface where the gel sticks has to be consequently chemically accessible by polymerization process while the other has to be chemically inert towards the radical polymerization. The supported gel can be then processed according to the traditional procedure for fixing and staining.

The resolution in the second dimension is increased as a consequence of the stacking effect during the equilibration Steps 6 and 7. Prior alkylation and SDS elektrokinetic equilibration together eliminate the need of treating the strip with equilibration solutions between first and second dimension. This means avoiding handling or moving the strip or closing the strip with valves, avoiding extra buffers, avoiding the use of coupling agarose or other stacking gel, reducing the complexity of operation, either manual or automatic, saving time, which means also minimized band broadening by diffusion, hence increased resolution also for the first separation. Finally, eventual washing out of proteins that can occur when using equilibration solutions is no longer an issue. The use of a gel formulation, which can be quickly polymerized and is stable as long as an external light source is not applied, avoids problems associated with gel preparation, avoids the need of prepolymerizing the gel before IEF and separate it from the strip by means of barriers, avoids otherwise long waiting times with consequently loss of resolution within the strip.

The object of the present invention is to extend the scope of the above described process steps according to EP 05007912.8 application and adding value to it.

The method previously disclosed is indeed particularly suitable to a series of new embodiments, achieving more homogeneous conditions for more gels in parallel, thus providing a more robust and convenient approach for more reproducible comparative studies; and all this still in a disposable format with reduced costs and minimum hands-on-time.

According to the present invention, an arrangement for the separation of a sample mixture for analytical reason based on two-dimensional gel electrophoresis is proposed, characterised in that at least two gel strips for the first separation step and corresponding gels for the second dimension are arranged and respectively at least two analytical processes can be executed in parallel.

According to one possible embodiment, at least two parallel first gel strips are preferably symmetrically arranged on a single carrier either on the same side or on opposite sides.

Further possible embodiments of the inventive arrangement are either described within the dependent claims or are described in more details within the following examples with reference to the attached drawings.

Description of the invention

Below, new inventive concepts, illustrated by schematic drawings, and for simplicity referred to as "mirror gels", "symmetric gels" and "parallel gels", the latter extendable to three dimensional analysis, are disclosed and the relative advantages highlighted. The description of the invention is therefore mainly a description of new embodiments, stressing the point on the fact that the method at the basis is the same previously derived and disclosed in EP 05007912.8 application. Thus no mention will be now made for e.g. the way in which sample loading, rehydration, IEF, equilibration and second dimension gel electrophoresis are carried out, because already described elsewhere or above. Part of the invention is however also another new method derived from the first, by which two-dimensional IEF can be performed without the use of valves as a prefractionation step before coupling to third-dimension gel electrophoresis.

Fig. 8 shows one possible embodiment as an example of the present invention in the form of so called "mirror gels".

This embodiment as shown in fig. 8 has a central or middle foil 31 in sandwich arrangement 33 between two external cover surfaces 35 and 37, the distance between which can vary so that the distance between each of these surfaces and the foil in the middle can assume the values e.g. of either ca. 0.7 mm or ca. 1 mm depending on applied external pressure or on a system of clamps 39 and 41 thanks to a system of compressible gaskets 43 and 45 as previously disclosed within the previous European application EP 05007912.8 , positioned this time at each side of the foil 31. The electrophoresis cassette-like arrangement 33 thus formed, including also buffer reservoirs 47 and 49 as illustrated in the drawing of fig. 8, can be injection moulded using light and / or UV transparent material and be all or partly disposable. The light or UV transparency is important due to the reason that for the formation of the gel for the separation in the second dimension a UV or light activated polymerisation is used which takes place within the two chambers 32 and 34 formed between the central foil 31 and the two cover surfaces 35 and 37.

Two identical first gel strips or IPG strips 51 are attached and aligned at a preferably precise position in correspondence of each other on either side of the central foil 31, a characteristic of which is to have gel bond properties on both sides. These first gel strips are preferably made out of a hydrophilic gel material as described and proposed within step 2 of the above disclosed step 2 in line with the EP 05007912.8 . In mirror configuration are also slits 53 on the external plates 35 and 37 at the gel / buffer interfaces, for which the same reasoning can be applied as also previously disclosed e.g. concerning the use of membranes. Another feature of the foil is a series of holes 55 and 57 along two lines parallel to the strips and at the two very extremities of the gel mold with the function to allow liquid communication between the two chambers 32 and 34 at each side of the foil. In this way the two chambers 32 and 34 can be filled simultaneously and homogeneously upon casting the gel solution and polymerisation accomplished also simultaneously by shortly shining e.g. UV light of appropriate wavelength and power from both sides as proposed within step 5 as mentioned above; and this is the same advantageous also when casting gradient gels.

The embodiment as shown in fig. 8, beside all the advantages of the general method, allows running the two separation steps of two-dimensional electrophoresis for two protein samples to be compared under nearly identical conditions as if they were run in a single gel like in the DIGE approach but without the need for pre labeling. Protein spots will theoretically form 2D mirror patterns on both sides of the transparent foil 31 through which they can then be directly compared after visual staining. More experiments could be made for control with different combinations of the two samples e.g. sample 1 versus sample 1, sample 2 versus sample 2 beside of course sample 1 versus sample 2.

A further embodiment as a further possible example according to the present invention is shown in figures 9a and 9b in the form of the so called "symmetric gels".

Fig. 9a shows the arrangement according to the present invention in a longitudinal section during the separation of the sample in the first and the second dimension and fig. 9b shows the gel strips after finishing the separation for analytical reason with the possibility to compare simultaneously the separation of two samples.

The embodiment 61, illustrated in fig. 9a has a foil 63 with doubled area and gel bonding properties only on one side. Two first gel strips 65 are now attached and aligned parallel at a certain distance between them and equally distant from the central axis A of the gel device 61 which now assumes a perfectly symmetrical shape. There are now three different buffer reservoirs 67, 69, and 71 for two gels, to anodes and one common cathode, integrated again in an injection moulded preferably UV transparent disposable body 64. In alternative said reservoirs could be also external, integrated instead in the instrument and the buffer could be made to recirculate. The distance between foil 63 and the inner surface 62 of the disposable body 64 can vary also in this case during the different steps due to the arrangement of compressible gaskets 68. The arrows 73 in fig. 9a indicate the direction of movement of the SDS during electro kinetic equilibration through the central slit as proposed in step 6 as disclosed above and the opposite direction of the second dimension run within the two chambers 66 for the two gels in the second dimension, after which the foil 63 can be peeled off, stained and bent over the midline equidistant from the two strips in order to achieve again in transparency easy and direct comparison, as shown in fig. 9b. This can be achieved e.g. by arranging a groove or recess 72 in the middle of foil 63.

Since homogeneous conditions can be guaranteed also in this case, the advantages are virtually the same as above. One unfavourable difference is the fact, that this embodiment is not suitable for casting gradient gels. One favourable difference is perhaps a simpler design with even lower cost of manufacture, certainly lower than for two separate single gel disposables.

Again a further embodiment as a further example according to the present invention is shown in fig. 10 in the form of the so called "parallel gels".

With this embodiment a number of first gel strips 81 (≥2) is arranged parallel equally distant and perfectly aligned with each other on a flat and thin carrier 83 trough which a series of slits 85 also parallel to the strips and equally distant from each strip are also arranged at the interface with the external buffer (not shown). The strips can be either identical to run different samples or replicas of the same samples in parallel or can carry different pH ranges for zoomed separations after e.g. a prefractionation step. At the perimeter of the strips carrier use is made again of compressible caskets 88 on top of which e.g. a solid block 87 is clamped consisting of parallel vertical gel moulds 89 (e.g. 1 mm spacing) for arranging the second gels for the separation in the second dimension, equally spaced to stand in correspondence of the first gel strips 81 underneath. The space in between is represented by cavities to be filled with the anodic running buffer until covering the gel moulds. Although this is a single piece block injection moulded once again with light or UV transparent material, features are inserted along the edges of the buffer cavities so that the block can be easily disassembled at the end of the analysis by weak manual torsion allowing opening of the individual gel moulds 89 for gel removal. The arrows 91 in the fig. indicated again the shape of the electric field, the direction of movement of the SDS during electrokinetic equilibration with stacking towards the center of each strip and subsequent protein transfer and migration into the vertical gels. Again reference is made to the previously described steps in relation to the prior European application EP 05007912.8 .

Again the main advantage over individual single gel analyses is to have more homogeneous conditions for several gels simultaneously so that higher reproducibility can be expected between them. Also remarkable reduction in the complexity of the system and operation is obtained compared to the handling of single strips and single gels, providing a really convenient straight forward approach for coupling strips in a tray to the second dimension separation. It should be noted also that this embodiment according to fig. 10 is amendable also to the classical method of running IEF, e.g. under mineral oil and to equilibrate the strips with standard equilibration solutions between first and second dimension.

Finally a further embodiment allows extension from two- to three-dimensional analyses as shown in fig. 11a and 11b. In contrast to the preceding embodiments in the arrangement as shown in figures 11a and 11b not a plurality of sample fractioning is possible or proposed, that any improved fractioning to achieve a far better resolution in the separation of a sample such as e.g. a protein sample.

Figure 11a shows the embodiment for the first dimension separation in cross sectional view and figure 11 b shows the embodiment for the first separation in view from above.

The fact that the strip rehydration and focusing can occur without any valves or barrier means makes it possible to couple different strips passing, e.g. from a broad pH range IPG strip to narrow pH range IPG strips as in figure 11. It is indeed possible to run IEF for the first strip 103 long enough to allow prefocusing and protein distribution along the pH range, but short enough for the proteins to be still charged before reaching their isoelectric points. In this way, after coupling to the parallel strips e.g. 104 - 111 ordered according to a pH range ladder, proteins can be transferred and separated orthogonally, thus achieving fractionation in the first dimension and increased resolution in the second dimension.

Thanks to faster polymerization and preferably to SDS electrokinectic equilibration, the embodiment as shown in figure 11a and 11b can therefore be now combined with that of figure 10 where the various individual gel moulds as designated with the referential number 89 are being arranged each above one of the parallel strips 104 to 111 and the separation in the third (before second) dimension can be carried out.

The embodiments as shown in fig. 8 till 11 are only examples for the description and the better understanding of the present invention. Compared with the gel electrophoresis disposables as proposed within the prior European patent application EP 05007912.8 , according to the present invention at the same time two or more separations of analytical mixtures are possible due to the fact, that within the EP 05007912.8 a simpler and better electrophoresis separation method to be automated is proposed using disposable bodies without the need of the arrangement of valves.

The present invention is of course not at all limited to the embodiments as shown in figures 8 to 11 and any development based upon the EP 05007912.8 proposing the use of at least two first gel strips and respectively having the possibility of executing in parallel at least two separation processes for at least two samples using electrophoresis is falling under the scope of the present invention.

References

  1. [1] Unlu M, Morgan ME, Minden JS. 1997. Difference gel electrophoresis: A single gel method for detecting changes in protein extracts. Electrophoresis 18:2071-2077 .
  2. [2] Sebastiano et al. Rapid Commun. Mass Spectrom. 2003, 17, 2380-2386 .


Anspruch[en]
Arrangement for the separation of a complex protein sample based on two-dimensional gel electrophoresis, the separation involving a first separation in a first-dimension strip on the basis of isoelectric points and a second separation in a second-dimension gel on the basis of molecular size characterised in that, at least two gel strips for the first separation step and corresponding gels for the second dimension are arranged on a single carrier either on the same side or on opposite sides and at least two analytical processes can be executed in parallel. Arrangement according to claim 1 characterized in, that the at least two gel strips for the first separation step are arranged parallel and/or symmetrically on the carrier. Arrangement according to one of the claims 1 or 2 characterised in that, above the at least two first gel strips at least one opposite disposable surface is arranged and that above each of the at least two first gel strips before introduction of the sample mixture to be separated a gap between the strip and the opposite surface, is provided, where such opposite surface at least at the sides of the gap is consisting of or coated with a hydrophobic or non gel sticking material and sample introduction occurs by hydrophilic guiding. Arrangement according to one of the claims 1 - 3, characterised in that, the disposable body of the arrangement is consisting of one carrier or bottom plate and at least one cover plate which can be moved such that the distance between the carrier or the bottom plate and the cover plate is variable. Arrangement according to one of the claims 1 - 4 characterized in that, elastic compressible frames are arranged, e.g. in the form of o-ring-like sealing, between the carrier or bottom plate and at least one cover plate, for movement of the carrier or bottom plate relative to the at least one cover plate to change the distance between the carrier or the plate and the cover plate and / or the distance between the first gel strips on the carrier or bottom plate and the respective opposite surface(-s). Arrangement according to one of the claims 1 - 5 characterized in that, any of the cover plates being arranged opposite to the first gel strips and preferably also the carrier is UV and visible-light transparent such that UV gel polymerization of the gel for the second dimension separation and image comparison of the separated protein samples are possible, the first gel strips and the second dimension gels being arranged respectively in contact with each other. Arrangement as according to one of the claims 1-6 characterized in that, no valves are arranged around the gel strips. Arrangement according to one of the claims 1 - 7 characterised in that, the arrangement is sandwich or mirror-like comprising a central carrier or plate on which on both sides at least one first gel strip is arranged, and each side is closed at a variable distance by a cover surface parallel to the plane of the central carrier. Arrangement according to claim 8 characterized in that, the central carrier is a foil with gel-bond properties on both sides. Arrangement according to claim 8 characterised in that, the spaces between the central carrier and the two opposite cover surfaces are establishing at least partially interspace like chambers for the formation of the second gels provided for the second dimension separation. Arrangement according to one of the claims 8 - 10, characterised in that, between the central carrier and the two cover surfaces elastic compressible frames are arranged, e.g. in the form of compressible o-ring-like sealings to allow distance variation of the two cover surfaces relative to the central carrier and / or the distance between the first gel strips and the respective opposite cover surfaces. Arrangement according to one of the claims 8 - 11 characterised in that, the sandwich or mirror-like arrangement is cassette-like comprising clamp-like means holding the two opposite cover plates in position and at the desired distance relative to the central carrier, the coverplates also comprising holes, e.g. for the introduction of the second gel formulating solution, etc., slits at the gel/buffer interfaces, and preferably buffer reservoirs. Arrangement according to one of the claims 8 -12 characterized in that, a series of holes or apertures is located at appropriate positions along the central carrier to allow liquid communication between the two interspace-like chambers. Arrangement according to one of the claims 1 - 7 comprising one foil or film like carrier with doubled area and gel bond properties on one side and with two first gel strips attached and aligned at a certain distance between them and preferably equally distant from a central axis of the film or foil like carrier, the carrier with the two first gel strips being closed by a cover surface of the disposable body, the distance between the carrier and the cover surface being variable during the different steps of the electrophoresis process due to e.g. an arrangement of compressible gaskets such as e.g. an o-ring like sealing between carrier and cover surface. Arrangement according to claim 14 characterised in that, the film or foil-like carrier is transparent and is comprising in the central area recess or groove like means such that, after the second dimension separation the film or foil-like carrier being peeled off the disposable body, can be bent and folded over the midline equidistant from the two first gel strips in order to achieve in transparency easy and direct comparison of the separated samples. Arrangement according to claim 14 or 15 characterized in that, the cover surface of the disposable body comprises holes, e.g. for the introduction of the second gel formulating solution, etc., slits at the gel/buffer interfaces, and preferably three buffer reservoirs, one central anodic reservoir and two opposing cathodic reservoirs, with the SDS electrokinetic equilibration for the two strips and the two second-dimension separations proceeding in opposite directions. Arrangement according to one of the claims 1 - 7 comprising a plurality of first gel strips arranged parallel preferably equally distant and preferably perfectly aligned with each other on one side of a plate or foil-like flat and thin carrier through which a series of slits also parallel to the first gel strips and preferably equally distant from each strip are arranged. Arrangement according to claim 17 characterised in that, in the area of the perimeter of the first gel strips plate or foil-like carrier use is made of a compressible gasket, on top of which a solid block of the disposable body is attached comprising parallel vertical gel moulds provided for the second dimension gel separations equally spaced to stand in correspondence of the first gel strips underneath, the gel moulds being made of a transparent material, and the distance of the solid block being variable relative to the plate or foil-like carrier. Arrangement according to claim 18 characterised in, that between the gel moulds means in form of grooves or the like are arranged so that the gel moulds each individually can easily be disassembled from the disposable body at the end of the second dimension separation step. Arrangement according to claim 17 further comprising a flat sealing cover at appropriate distance relative to the plate or foil-like carrier until the first dimension separations are completed and being then substituted with a solid block according to claim 18 before proceeding with gel casting, polymerization and second dimension separations. Arrangement optionally according to one of the claims 1 to 7 for the prefractionation of a complex protein sample, characterized in that, on plate or foil-like carrier one primary first gel strip is attached, provided for a first quick separation of the sample in a broad pH range such that, the proteins of the sample are still charged before reaching their isoelectric points, the carrier further comprising a plurality of secondary first gel strips being arranged in contact at one side with and nearly perpendicular to the primary gel strip, and nearly parallel between themselves, that is at equivalent distances between each other, said secondary gel strips representing a pH ladder each corresponding to a narrow pH range comprised within the broad pH range of the primary strip, so that transfer of zones from the primary strip to the respective secondary strips can occur and increased resolution can be obtained. Arrangement resulting from the combination of the solid block of gel moulds according to any one of the claims 18 to 20 with the arrangement according to claim 21. Process for the separation of a complex protein sample based on two dimensional gel electrophoresis, the separation involving a first separation in the first dimension strip on the basis of isoelectric points and a second separation in a second dimension gel on the basis of molecular size, characterized in that, within at least two gel strips, being arranged on a single carrier either on the same side or on opposite sides, and corresponding second-dimension gels, at least two analytical processes are respectively being executed in parallel, the second dimension gels being in contact with the first dimension strips. Process according to claim 23, characterized in that, after the first dimension separation the distance between the carrier and the at least one opposite disposable surface is being increased to provide optimal conditions for gel casting, polymerization, SDS equilibration, transfer and separation into the second-dimension gel. Process according to one of the claims 23 or 24 characterized in that, the analytical processes are based on alkylation prior to sample introduction and strip rehydration, hydrophilic sample guiding, fast UV gel polymerization for the production of the second-dimension gels and on SDS electrokinetic equilibration of the sample, fractioned in the first dimension before entering into the second-dimension gel. Use of the arrangement according to one of the claims 1 - 22 for the separation of a sample mixture such as in particular of a complex protein sample.






IPC
A Täglicher Lebensbedarf
B Arbeitsverfahren; Transportieren
C Chemie; Hüttenwesen
D Textilien; Papier
E Bauwesen; Erdbohren; Bergbau
F Maschinenbau; Beleuchtung; Heizung; Waffen; Sprengen
G Physik
H Elektrotechnik

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