WO2004011926A1

WO2004011926A1 - Method of separating a mixture of chemical compounds and support for performing separation

Info

Publication number: WO2004011926A1
Application number: PCT/GB2003/003215
Authority: WO
Inventors: Richard Owen
Original assignee: Pa Consulting Services Ltd
Priority date: 2002-07-29
Filing date: 2003-07-29
Publication date: 2004-02-05
Also published as: GB0317726D0; AU2003281728A1; GB2393256B; GB0217481D0; GB2393256A

Abstract

A method of separating a mixture of charged chemical compounds comprises the steps of: (a) providing an electrophoretic separation medium (1) which carries (i) the mixture of charged compounds at a first region (4) of the medium, and (ii) a staining agent for the charged compounds at a second region (5) of the medium which is spaced from said first region, (b) electrophoretically migrating the staining agent towards the charged compounds, to bind the staining agent to the charged compounds and to reduce the concentration of staining agent at said second region, and(c) electrophoretically separating the charged and stained compounds.

Description

METHOD OF SEPARATING A MIXTURE OF CHEMICAL COMPOUNDS AND SUPPORT FOR PERFORMING SEPARATION

Field of the Invention

The present invention relates to methods of separating a mixture of chemical compounds, and to supports for performing separation of mixtures of chemical compounds.

Background to the Invention

Electrophoresis is a technique which allows a mixture of charged chemical compounds to be separated by virtue of their sizes and/or charges. The technique is commonly applied to separate mixtures of large numbers (up to thousands) of different proteins or nucleic acids.

In a typical protein molecular weight separation process, the proteins are mixed with sodium dodecyl sulphate (SDS), and the protein/SDS mixture is located at one end of a gel separation matrix or medium (formed e.g. of polyacrylamide) . The SDS denatures the proteins and acts as a charging agent for the proteins by binding to each protein in an amount which is proportional to the size of the protein molecule, thereby providing the protein with a negative electrical charge in proportion to its size.

Buffer solutions are then positioned at opposing ends of the separation matrix, with a cathode contacting one buffer solution and an anode contacting the other. The charged proteins are usually placed in a well adjacent the cathode buffer. When an electric field is applied between the cathode and the anode, the charged proteins- migrate towards to the anode at rates which depend on their respective sizes, larger proteins being slower than, smaller proteins. Thus the proteins separate as they pass through the gel, with the result that the position of a particular protein in the gel correlates to the size of that protein.

For mixtures of nucleic acids a separate charging agent is not required because the phosphate group associated with each nucleotide already has a negative charge. Because of this, the overall charge carried by each nucleic acid is proportional to the number of phosphate groups of the acid and hence to its size.

A critical stage of the electrophoresis procedure is the detection or visualisation of the nucleic acids/proteins in the gel. Without visualisation, the separation pattern cannot be seen, and the nucleic acids/proteins of interest are unlocatable and hence unidentifiable.

In respect of nucleic acid separation, visualisation is commonly accomplished by binding the fluorophore staining agent ethidium bromide to the nucleic acids. The ethidium bromide does not interfere with the electrophoresis and is typically pre-loaded into the gel. This advantageously allows the nucleic acids to be monitored as they migrate so that, if • ecessary, changes can be made to the procedure during separation.

For proteins, however, a problem arises in that the use of many common staining agents (e.g. silver^' stain, Coomassie™ Brilliant Blue, etc.) involves processing which can only be carried out after separation is complete. Typically, the gel is removed from the electrophoresis equipment, and fixed and soaked in a bath of chemical dye for a minimum of 30 minutes. Most dyes then require an extensive de-staining procedure which removes the dye from the empty portions of the gel while leaving some bound to the proteins present in the gel. Thus, post-separation processing is time-consuming, expensive, and not particularly user-friendly, and an alternative procedure would be desirable which allowed proteins to be visualised within the gel while electrophoresis progressed.

US 6,277,259 discloses a two-dimensional gel electrophoresis- based protein separation method for performing isoelectric focusing (IEF) in a first dimension followed by sodium dodecyl sulphate-polyacryla ide gel electrophoresis (SDS- PAGE) in a second dimension. During second dimension separation, the migrating proteins are stained "in migratio" by complexation with a complexing fluorophore incorporated directly into the separation medium. However, for this method to work, the total amount of fluorophore added to the gel has to be relatively high. This increases the background fluorescence of the gel and so decreases both the sensitivity of detection and the achievable resolution.

Alternatively, Molecular Probes™ (manufacturer of the Sypro™ range of fluorophore dyes, which are commonly used for protein staining) state in their MP 06650 product information publication (revised 15 January 2001) that Sypro dyes can be added- directly to the cathode buffer solution. A continuous sheet of dye flows out of the buffer reservoir and then moves through the gel with the SDS front so that all the proteins are stained. However, de-staining is still required to reduce background fluorescence, and Molecular Probes state that poorer protein staining results than standard post- staining methods.

A similar approach is disclosed in US 5,132,439 and G.A. Larson and J.W. Shultz, BioTechniques, Vol. 15, No. 2, 316- 323, (1993) . Both these documents propose diluting protein stain into the cathode buffer before electrophoresis. US 5,132,439 asserts that protein bands in lanes containing 2.0 and 1.0 μg of protein per band became visible during separation. However, for improved visibility of the bands, a post-separation destaining step was performed.

Two papers (Anal . Chem . , 2000, 72: 2519-2525 and Electrophoresis, 1998, 19: 2169-2174) describe similar pre- labelling methods in which Sypro dye is added to SDS-protein samples before the performance of electrophoretic separation. In neither case was post-run staining performed. The Anal . Chem . paper related to slab gel electrophoresis and the Electrophoresis paper related to capillary electrophoresis.

It was reported in the Anal . Chem . paper, however, that the Sypro dye preferentially binds to micelles of SDS in the sample and not to the protein/SDS complexes. Thus the Sypro dye had to be added immediately prior to sample loading to limit dye transfer from the protein/SDS complexes to the SDS micelles. Also, during electrophoresis the Sypro dye forms a front (with that part of the SDS which is not bound to the proteins) which runs ahead of the proteins. If too much dye is used this dye front can obscure the signal from nearby (relatively mobile) low molecular weight proteins, but if too little is used the proteins cannot be visualised properly.

Furthermore, the amount of SDS added to the sample has to be decreased (by a factor of 20 from 1% to 0.05%) to prevent the SDS from binding all the Sypro dye. However, if too little SDS is added, the mobilities of the proteins are affected. Thus, although pre-labelling can apparently work, it is highly sensitive to experimental conditions. Summary of the Invention

Consequently, it is an object of the present invention to provide a method of electrophoretically separating a mixture of chemical compounds (particularly proteins) which allows the compounds to be visualised during separation and which avoids some or all of the drawbacks mentioned above.

In general terms the present invention provides a method of electrophoretically separating a mixture of chemical compounds, in which the compounds and a staining agent for the compounds are provided at discrete locations on a separation medium. The compounds may be electrically charged or a charging agent for the compounds may be provided at a further discrete location on the medium. The staining agent (and optionally the charging agent) are then migrated towards the compounds to stain (and optionally charge) the compounds in si tu . The separation medium may be e.g. a slab gel or a capillary gel.

In a first aspect the invention provides a method of separating a mixture of charged chemical compounds, the method comprising the steps of:

(a) providing an electrophoretic separation medium which carries (i) the mixture of charged compounds at a first region of the medium, and (ii) a staining agent for the charged compounds at a second region of the medium which is spaced from said first region,

(b) electrophoretically migrating the staining agent towards the charged compounds to bind the staining agent to the charged compounds and to reduce the concentration of staining agent at said second region, and

(c) electrophoretically separating the charged and stained compounds . After step (c) the separated charged and stained compounds can be identified in a number of ways. For example, the staining pattern of the compounds may be characteristic of a known compound mixture which can be recognised by the user or by reference to a database of staining patterns.

Alternatively or additionally, the individual separated compounds may be characterised by analytical techniques, such as mass spectroscopy.

Typically the charged compounds will be proteins which have been charged by a charging agent such as SDS. However, they may also be compounds which are intrinsically charged, such as nucleic acids. The staining agent should also be charged so that it can electrophoretically migrate. For example, the staining agent may be a Sypro dye.

The staining of the charged compounds may be performed as an integral part of a separation run. For example, the staining agent is typically positioned between a field electrode (usually the cathode) and the charged compounds such that application of an electric field drives the staining agent towards the charged compounds, which will generally migrate much more slowly than the staining agent. In this way the staining agent catches up with and binds to the charged compounds which are thereby stained in situ. Any excess staining agent passes through the charged compounds and, because the staining agent migrates as a discrete region or pulse, it leaves little or no agent behind to provide unwanted background staining. This is in contrast to the methods described in US 6,277,259 and the Molecular Probes MP 06650 product information publication which require subsequent de-staining steps to remove or reduce background levels of staining. Thus in one embodiment, in step (b) , the staining agent electrophoretically migrates to bind to the staining agent and to leave behind substantially no staining agent at said second region. By "substantially no staining agent" we preferably mean an amount of staining agent such that the separation medium does not need a de-staining step in order for the separated charged and stained compounds to be visualised or otherwise identified.

Even though the second region of the separation medium may be much smaller than the first region, the staining agent can be present at a relatively high concentration in the second region which will lead to an efficient binding reaction with the charged compounds. Such a high concentration should not lead to undesirable levels of background staining, because the staining agent typically migrates as a discrete zone. That is, although a trail of residual staining agent may form behind by the zone of migrating staining agent to a limited extent, the concentration of staining agent in the trail should be sufficiently low to avoid the need for post- separation de-staining.

Furthermore, any excess staining agent migrates relatively quickly away from the charged and stained compounds in a discrete front. This front, one or more convenient control compounds from the compound mixture, and/or a reference compound positioned outside the compound mixture may be used as a marker (s) to monitor the overall speed of migration, allowing the experimental conditions to be controlled e.g. to speed up or slow down separation as required.

Thus preferably the method further comprises the step of: controlling the separation of the charged and stained compounds in real time by monitoring the migration of the staining agent, one or more of the charged and stained compounds, and/or a reference compound.

Control of the separation of the charged and stained compounds may be accomplished e.g. by changing the voltage, the current, the active cooling, or any other controllable parameter that affects the running of the separation process. This could be accomplished manually or by using an automatic control loop linked to real time monitoring of the electrophoretic migration. The benefit of this is that active steps can be taken during the run to minimise variations between separate gel runs (migration rate variation between nominally identical gels can be a problem with conventional separation techniques) .

Clearly, when the method is applied to chemical compounds which are charged by a charging agent (e.g. SDS-charged proteins) , the staining agent will be introduced to the compounds after they have been charged. This is advantageous because, compared with the pre-labelling methods described above in the Anal . Chem . and Electrophoresis. papers, precise determination of the amount of staining agent to be employed (and indeed precise determination of the amount of charging agent) is much less critical, competition between the staining agent and the chemical compounds for the charging agent being avoidable in the method of the present invention. For example, when an excess of charging agent is provided with the mixture of charged compounds at the first region of the separation medium, application of an electric field to migrate the staining agent to the charged compounds can simultaneously migrate the excess charging agent away from the charged compounds before the staining agent arrives thereat.

Thus, preferably the method further comprises the step of: removing excess charging agent from the charged compounds before the staining agent is bound to the charged compounds .

More preferably the excess charging agent is electrophoretically removed from the charged compounds. However, it may also be possible to render the charging agent unavailable for interaction with the staining agent without physically moving the agent from the vicinity of the compounds. Thus by "removing" we mean a process which either physically moves the charging agent away from the charged compounds or renders the charging agent unavailable for binding to the staining agent.

The method may be used in one dimensional or two dimensional (see e.g. US 6,277,259) electrophoretic separation techniques .

The separation medium of step (a) may be provided in a number of ways. For example, the separation medium may be preloaded with charging agent, and subsequently loaded with the compound mixture at the first region and the staining agent at the second region so that the mixture of charged compounds at the first region of the medium is formed in si tu . When the charging agent is SDS, the separation medium may be preloaded with SDS at a concentration below the critical micelle concentration for SDS such that the staining agent does not bind to the SDS before the staining agent arrives at the mixture of charged compounds. Alternatively, however, the charged compound mixture may be formed before it is loaded onto the separation medium at the first region.

Furthermore, the charged compound mixture and the staining agent may be sequentially loaded onto the separation medium at the same loading position, with the charged compound mixture being electrophoretically migrated away from the loading position towards the first region before the staining agent is loaded.

A further aspect of the invention provides a method of separating a mixture of chemical compounds, the method comprising the steps of:

(a) providing an electrophoretic separation medium which carries (i) the mixture of chemical compounds at a first region of the medium, (ii) a staining agent for the chemical compounds at a second region of the medium which is spaced from said first region, and (iii) a charging agent for the chemical compounds at a third region of the medium which is spaced from said first and second regions, (b) electrophoretically migrating the charging agent towards the chemical compounds to bind the charging agent to the chemical compounds and to reduce the concentration of charging agent at said third region,

(c) electrophoretically migrating the staining agent towards the charged compounds to bind the staining agent to the charged compounds and to reduce the concentration of staining agent at said second region, and

(d) electrophoretically separating the charged and stained compounds .

Thus the method of this aspect of the invention is similar to the method of first aspect except that charging agent is provided at a discrete region of the separation medium and binding of the charging agent to the chemical compounds is an integral part of the method. Thus charging, as well the staining, of the chemical compounds can be performed in si tu . Any one or combination of the optional features of the first aspect of the invention may also be applied to this aspect. Preferably the charging agent and the staining agent are positioned between a field electrode and the chemical compounds, with the charging agent closer to the compounds than the staining agent. In this way, on application of an electric field, the charging agent arrives first at the compounds, followed at an interval by the staining agent (charging agents such as SDS generally migrate at about the same speed as staining agents such as Sypro dyes) . Thus typically, by the time the staining agent reaches the chemical compounds they are already charged, and any excess charging agent has migrated as a discrete front beyond the compounds so that competition between the compounds and the charging agent for the staining agent can be avoided. Any excess staining agent may then also migrate as a discrete front beyond the compounds. It will not matter if this front subsequently catches up with the charging agent because the excess agents should then be far enough away from the chemical compounds such that charging and staining of the compounds is unaffected.

Advantageously, the charging agent can be present at a relatively high concentration in the third region, such that an efficient charging reaction with the compounds ensues. Also, because the staining agent migrates as a discrete zone, significant levels of residual charging agent forming a trail behind the migrating zone can be avoided. This is desirable, as such residual charging agent might otherwise bind with the staining agent before it reaches the compounds to reduce the level of "available" staining agent and increase background staining.

Further aspects of the invention relate to supports which may be used in the methods of the previous aspects. For example, in one aspect the present invention provides a support for performing electrophoretic separation of a mixture of charged chemical compounds, the support comprising an electrophoretic separation medium having (i) a first region for carrying the mixture of charged compounds, and

(ii) a second region which is spaced from said first region and which carries a staining agent for the charged compounds, the support being arranged such that, when said first region carries the mixture of charged compounds and an electric field is applied across the medium, the staining agent migrates towards the charged compounds to bind the staining agent to the charged compounds and to reduce the concentration of staining agent at said second region.

In one embodiment, the first region carries the mixture of charged compounds .

In- another aspect the present invention provides a support for performing electrophoretic separation of a mixture of chemical compounds, the support comprising an electrophoretic separation medium having (i) a first region for carrying the mixture of chemical compounds, (ii) a second region which is spaced from said first region and which carries a staining agent for the chemical compounds, and (iii) a third region which is spaced from said first and second regions and which is for carrying a charging agent for the chemical compounds, the support being arranged such that, when said first region carries the mixture of chemical compounds, said third region carries the charging agent, and an electric field is applied across the medium, (i) the charging agent migrates towards the chemical compounds to bind the charging agent to the chemical compounds and to reduce the concentration of charging agent at said third region, and (ii) the staining agent migrates towards the charged compounds to bind the staining agent to the charged compounds and to reduce the concentration of staining agent at said second region.

In one embodiment, the first region carries the mixture chemical compounds.

In another embodiment the third region carries the charging agent.

The charging agent of this or the previous aspect may be SDS. The staining agent may be a Sypro dye.

The present invention will now be described in relation to detailed embodiments and with reference to the following drawings in which:

Figs, la to h are a series of schematic drawings of a support for performing electrophoretic separation showing the sequence of events in a separation run,

Figs. 2a to c are a sequence of schematic concentration profiles for dye being migrated along a gel separation lane according to a method of the present invention,

Figs. 3a to c are a sequence of schematic concentration profiles for dye being migrated along a gel separation lane according to a conventional method,

Fig. 4 shows schematically an alternative support for performing electrophoretic separation,

Fig. 5 shows photographs of a gel strip (A) stained in si tu according to the present invention and (B) conventionally stained post-run, Fig. 6 shows (a) a gel generated according to the present invention and (b) a gel generated by conventional one hour post-run staining with Sypro Orange, in each case the migrated protein was relatively high concentration bovine serum albumin (BSA) , the numbers refer to the nanograms of BSA applied to each lane of the respective gel,

Fig. 7 shows (a) a gel generated according to the present invention and (b) a gel generated by conventional one hour post-run staining with Sypro Orange, in each case the migrated protein was relatively low concentration BSA, the numbers refer to the nanograms of BSA applied to each lane of the respective gel, and

Fig. 8 shows (a) linear-linear and (b) log-linear plots of counts from a Bio-Rad FX scanner at high sample intensity setting for detected BSA at concentrations of from 16 to 32000 ng/well.

Detailed Description

The present invention provides a novel way of adding a stain or label to an electrophoretic separation medium such that the stain binds to the compounds being separated during the electrophoresis run. Reacting the stain with the compounds during the run overcomes problems identified in the prior art .

The detailed embodiments described below relate to SDS-PAGE protein separation with Sypro fluorescent dye staining.

However, the invention has broader applicability and may be used in the electrophoretic separation of e.g. nucleic acids. Furthermore, the skilled person would recognise that other agents may be used in place of SDS and Sypro dyes. He would also recognise that other separation media and separation formats may be used. For example, for convenience, the embodiments described below relate to single separation lanes of polyacrylamide gel separation media. However, the invention may be applied to multi-lane separation, ID slab gel electrophoresis, 2D gel electrophoresis, capillary gel electrophoresis etc. The separation media may be formed from other substances such as agar, agarose, sepharose, cellulose and gelatin.

Figs, la to h are a series of schematic drawings showing the sequence of events of an electrophoretic separation run according to the present invention. Fig. la shows a support comprising a lane 1 of polyacrylamide gel which at one end 2 is contacted to a cathode via a cathode buffer and at the other end 3 is contacted to an anode via an anode buffer.

A sample well 4 (defining a first region of the separation medium) is formed in the lane close to the cathode buffer and is loaded with a protein/SDS mixture indicated by grey shading. The SDS binds to the proteins, but as the SDS is present in excess, there is a mix of free SDS and protein/SDS complexes in the well.

A line of Sypro dye 5 (defining a second region of the separation medium spaced from the first region) is positioned between the cathode buffer and the sample well.

When an electric field is applied between the electrodes the dye, free SDS and protein/SDS complexes all start to migrate towards the anode. However, the dye and free SDS (which move at about the same speed) migrate much faster than the relatively large protein/SDS complexes so that when the dye reaches the sample well, the free SDS has already advanced down the separation lane towards the anode whereas the protein/SDS complexes have only just exited from the sample well and have not yet significantly separated out into bands of differently sized proteins. This is the situation shown in Fig. lb, with the dye indicated by a dashed line, the free SDS by a dotted line and the protein/SDS complexes by a continuous line.

In Fig. lc the dye enters the sample well (as indicated by grey bands) and in Fig. Id almost catches up with the protein/SDS complexes which have still not progressed far along the separation lane. In Fig. le the dye reaches the protein/SDS complexes and binds with them to form protein/SDS/dye complexes. These are indicated in subsequent drawings as paired dashed/continuous lines. The free SDS is sufficiently far from the protein/SDS/dye complexes at this time such that it is not exposed to the dye. The excess dye (dashed line) , then passes through the protein/SDS/dye complexes and continues to migrate at its initial rate behind the excess SDS but in advance of the complexes.

In Figs. If to h the excess SDS and excess dye progress down the separation lane and the excess SDS eventually runs into the anode buffer. The run is stopped when the dye is detected by a fluorescence detector (not shown) at the end of the gel. Meanwhile the protein/SDS/dye complexes resolve into separate bands corresponding to differently sized proteins, and in Fig. lh are spread out along the lane with the smallest proteins closest the anode and the largest proteins closest the cathode. The gel now contains some free dye proximate the anode, dye in protein/SDS/dye complexes and insignificant levels of background dye in the gel. Thus the gel does not need de-staining and the proteins can be visualised immediately by viewing in UV light and photographed if required. It is also possible to position a fluorescent detector over the gel so that the proteins can be viewed as they migrate. This allows the proteins to be monitored during the run and the hence enables the use of marker proteins to control the voltage between the electrodes to decrease run-to-run variation. This control can be automated if desired.

A significant feature of the procedure is the discrete and localised nature of the initial line of dye 5. The dye is highly concentrated in the line such that it produces an efficient staining reaction. However, it and the excess charging agent also efficiently vacate their initial and subsequent positions as the run progresses so that a high background level of dye is avoided.

This is illustrated in Figs. 2a to c, which show schematically the dye concentration profile along a gel separation lane during the course of a run. The protein/SDS mixture is first loaded into the sample well and migrated a short distance along the lane. Dye is then loaded into the sample well, the dye being spaced from the protein/SDS mixture by this short distance. This is the situation shown in Fig. 2a, peak 20 being the concentration of dye in the sample well. As separation proceeds (Fig. 2b), the dye migrates down the lane as a narrow, high concentration peak 21. Little or no dye is left behind, except, of course, bands 22 formed when the dye catches up and binds with separated proteins from the protein/SDS mixture. Finally, as shown in Fig. 2c, the dye reaches and is diluted in the anode reservoir, leaving essentially only bands 22 behind to mark the positions of separated proteins.

In contrast, Figs. 3a to c show schematically the dye concentration profile along a gel separation lane during the course of a run performed according to the conventional method of e.g. US 5,132,439. Here the dye is loaded into the cathode reservoir, as shown in Fig. 3a. As separation proceeds (Fig. 3b) the dye in the reservoir moves evenly along the length of the entire lane, binding to proteins as it passes through them to form bands 23. Finally, as shown in Fig. 3c, the dye reaches and runs off into in the anode reservoir, leaving bands 23 behind to mark the positions of separated proteins. However, the gel is also saturated with dye so that the bands 23 are smaller (and hence less visible) relative to the background dye concentration than the bands 22 formed by the procedure illustrated by Fig 2.

The dye can be applied to the gel in a number of ways. For example a second well could be added between the cathode and the sample well for receiving the dye. Alternatively the dye could be incorporated into a pre-cast gel. The dye could also be applied onto filter paper and laid across the gel just prior to use. Whichever application method is selected, however, it is important to prevent SDS leaking to the dye, as the dye would then bind to the free SDS leaving none to bind to the protein/SDS complexes.

Fig. 4 shows schematically an alternative support for performing protein mixture electrophoretic separation using SDS and Sypro dye. Like the support of Figs, la-h, the support has a lane 11 of polyacrylamide gel which contacts a cathode via a cathode buffer at one end 12 and an anode via an anode buffer at the other end 13. However, in this case, the lane has a sample well 14 which holds a protein mixture, a line of Sypro dye 15 positioned between the cathode buffer and the sample well 14, and a well 16 (defining a third region of the separation medium) containing SDS positioned between the dye 15 and the sample well 14. With this arrangement, when an electric field is applied between the electrodes, the SDS and Sypro simultaneously migrate at about the same speed towards the protein mixture, which is initially uncharged and so remains in the sample well. The SDS leaving well 16 is highly concentrated such that when it reaches the sample well it produces an efficient charging reaction with the proteins to form protein/SDS complexes. The protein/SDS complexes then start to migrate with excess SDS moving in a front before them.

Subsequently, the separation run continues in the same way as the separation^' run already described in respect of the support of Figs, la to h.

A significant advantage compared with the pre-labelling methods described in the Anal . Chem . and Electrophoresis papers is that the amount of SDS in the sample does not have to be reduced (the standard 1% can be used) . Thus protein mobility is not affected and more protein in the sample enters the gel.

Furthermore, the amount of dye does not need to be accurately titrated and the timing of the dye addition to the gel is not critical. Indeed, the dye can be incorporated in the gel when the gel is manufactured. Although, the amount of dye used is relatively low so that background staining is reduced, the high concentration of dye in the dye front produces an efficient staining reaction. Also the amount of protein in the sample does not need to be accurately controlled.

The gel does not require post-run staining or de-staining, which significantly adds to the convenience and speed of the process. Also the proteins can be visualised during the electrophoresis run so that changes can be made to the run by the user in real time to improve the separation of particular proteins .

Example 1

An aqueous gel formulation containing 10% acrylamide, 0.05% SDS, 150 mM Tris, 150 mM Tricine pH 8.1, 1/1000 (w/v) ammonium persulphate, 1/2000 (v/v) TEMED was prepared. The formulation was used to produce a 60 mm long and 1 mm² cross- sectional area gel strip. The strip was arranged with one end contacting a cathode buffer and the other end a anode buffer.

A protein sample containing 0.85 mg/mL of protein mixture, 5% 2-mercaptoethanol, 1% SDS and 2mM Tris pH 8.1 was prepared and boiled for 3 minutes before the separation run. The protein mixture used Beckman™ native protein molecular weight markers for SDS-PAGE having protein sizes of 205, 116, 97, 66, 45, 29 and 14.2 kD

2 μL of the protein sample was loaded into a first slot cut in gel strip near the cathode buffer. 4 μL of Sypro red diluted 1 to 250 in water was loaded in a second slot cut into the gel between the first slot and the cathode buffer.

A 400 V potential was then applied between the ends of the gel strip to initiate electrophoresis. The separation run took 15 minutes to complete, with the current varying in the range 3 to 5 rriA.

The Sypro dye stained the protein/SDS complexes in situ during the run. Photograph A of Fig. 5 shows the gel strip after the run. Bands corresponding to all the proteins in the protein mixture are clearly visible. The strip was then stained using a conventional post-run staining procedure and re-photographed to provide photograph B of Fig. 5. This shows that the staining pattern produced by the in si tu staining is the same as that produced by conventional post-run staining.

Staining Reagents, Protocol and Tolerance Ranges

We have performed protein separation and staining according to the present invention using the following reagents and in situ staining (ISS) protocol.

Protein sample buffer:

5% (v/v) mercaptoethanol, 1% (w/v) SDS, 4% (w/v) sucrose, lOOmM Tris-HCl pH 8.3 + a few grains of bromophenol blue. Add protein to around 0.2mg/mL of buffer and boil it for 3 minutes .

Electrophoresis buffer: 25mM Tris, 192mM Glycine pH 8.3 with 0.02% (w/v) SDS.

Staining buffer:

Stock Sypro Orange diluted 1/200 in 78mM Tris, 600mM Glycine pH 8.3, 0.4% (w/v) SDS.

Gel:

Standard pre-poured Bio-Rad Ready gels. 12% acrylamide,

Tris/HCl pH (no SDS), 30μL sample volume.

ISS Protocol:

- Load gels with around lOμL protein sample in each well.

- Run gel at 150V for around 5 minutes.

- Stop run, wash the wells with electrophoresis buffer to remove sucrose. - Add 15μl staining buffer to each well. - Continue the run at 150V for another 40 min or until the blue line (from the bromophenol blue) runs off the bottom of the gel.

- Extract gel and view on UV transilluminator or using scanning equipment e.g. Bio-Rad' s FX scanner.

Tolerance ranges for the staining buffer:

(i) The stock Sypro Orange can be diluted 1/10,000 - 1/200. Too little Sypro Orange and the proteins may not be visualisable . Too much and the Sypro may not migrate cleanly down the gel - residues can be left in the gel that cause background fluorescence.

(ii) Preferably, the ratio between the Tris and the Glycine should be maintained, but the absolute concentration can be reduced slightly. If the absolute concentration is too low (less than about 25mM Tris and 192mM Glycine) or too high (greater than about 156mM Tris and 1200mM Glycine) the protein bands can distort as the Sypro band passes through them. (iii) The amount of SDS can be increased slightly to around 0.5% - if it is too high the Sypro may not transfer to the protein bands. If it is too low, i.e. below about 0.01%, the Sypro may not be able to enter the gel and migrate through ^■ it.

Tolerance ranges for the electrophoresis buffer: The electrophoresis buffer is a standard Tris/Glycine solution. However, the SDS concentration is reduced from 0.5% (w/v) (recommended by the manufacturers of the Sypro dyes) to 0.02% (w/v). If no SDS is added to the electrophoresis buffer, the Sypro may not be able to migrate properly through the gel. If 0.05% SDS is added (i.e. above the effective critical micelle concentration) , the Sypro may be removed from the proteins by the SDS micelles that enter and move through the gel. Figs. 6a and b show a comparison between (a) a gel generated according to the above protocol and (b) a gel generated by conventional one hour post-run staining with Sypro Orange. Both gels were imaged using a Bio-Rad FX scanner at high sample intensity setting. The migrated protein was bovine serum albumin (BSA) . The numbers refer to the nanograms of BSA applied to each lane of the respective gel.

Figs. 7a and b show a similar comparison (a) a gel generated according to the above ISS protocol and (b) a gel generated by conventional one hour post-run staining with Sypro Orange. Both gels were imaged using the Bio-Rad FX scanner at low sample intensity setting. Again the migrated protein was BSA, but this time at lower concentrations.

Figs. 6 and 7 show the ISS protocol can lead to improved results compared with conventional techniques. This is further illustrated by the following table which compares the signal to noise (S/N) ratios obtained for the ISS protocol at high BSA concentrations with the corresponding S/N ratios for the conventional post-run staining technique.

At low concentrations the ISS protocol gave a detection limit of 4 ng BSA. We were unable to match this with the conventional post-run staining technique.

The ISS protocol also allowed detection with good linearity over three orders of magnitude of BSA concentration. Figs. 8a and b respectively show linear-linear and log-linear plots of counts from the Bio-Rad FX scanner at high sample intensity setting for BSA detected using the ISS protocol at concentrations of from 16 to 32000 ng/well.

While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.

Claims

1. A method of separating a mixture of charged chemical compounds, the method comprising the steps of: (a) providing an electrophoretic separation medium which carries (i) the mixture of charged compounds at a first region of the medium, and (ii) a staining agent for the charged compounds at a second region of the medium which is spaced from said first region, (b) electrophoretically migrating the staining agent towards the charged compounds, to bind the staining agent to the charged compounds and to reduce the concentration of staining agent at said second region, and

(c) electrophoretically separating the charged and stained compounds .

2. A method of separation according to claim 1, wherein the mixture of charged compounds is a mixture of chemical compounds which are charged by a charging agent.

3. A method of separating a mixture of chemical compounds, the method comprising the steps of:

(d) electrophoretically separating the charged and stained compounds.

4. A method of separation according to claim 2 or 3, further comprising the step of: removing excess charging agent from the charged compounds before the staining agent is bound to the charged compounds.

5. A method of separation according to claim 4, wherein the excess charging agent is electrophoretically removed from the charged compounds .

6. A method of separation according to any one of claims 2 to 5, wherein the charging agent is sodium dodecyl sulphate (SDS) .

7. A method of separation according to any one of the previous claims further comprising the step of: controlling the separation of the charged and stained compounds in real time by monitoring the migration of the staining agent, one or more of the charged and stained compounds, and/or a reference compound.

8. A support for performing electrophoretic separation of a mixture of charged chemical compounds, the support comprising an electrophoretic separation medium having (i) a first region for carrying the mixture of charged compounds, and

9. A support according to claim 8, wherein said first region carries the mixture of charged compounds.

10. A support for performing electrophoretic separation of a mixture of chemical compounds, the support comprising an electrophoretic separation medium having (i) a first region for carrying the mixture of chemical compounds, (ii) a second region which is spaced from said first region and which carries a staining agent for the chemical compounds, and (iii) a third region which is spaced from said first and second regions and which is for carrying a charging agent for the chemical compounds, the support being arranged such that, when said first region carries the mixture of chemical compounds, said third region carries the charging agent, and an electric field is applied across the medium, (i) the charging agent migrates towards the chemical compounds to bind the charging agent to the chemical compounds and to reduce the concentration of charging agent at said third region, and (ii) the staining agent migrates towards the charged compounds to bind the staining agent to the charged compounds and to reduce the concentration of staining agent at said second region.

11. A support according to claim 10, wherein said first region carries the mixture of chemical compounds.

12. A support according to claim 10 or 11, wherein said third region carries the charging agent.

13. A support according to any one of claims 8 to 12, wherein the charging agent is SDS_: