Abstract:
An improved system for running electrophoresis gels “face up” is described. The system includes a single gel capacity strip holder, two electrodes, a sample cup, and a cover. The system provides a means of separating basic proteins on any length gel strip and the cup provides a user-friendly means of successfully putting the proteins on the gel face.

Description:
FIELD OF THE INVENTION 
     The present invention relates to equipment and methods used in complex protein mixture analysis by 2-dimensional electrophoresis. 
     BACKGROUND OF THE INVENTION 
     For any type of detailed substance analysis, a homogeneous sample of the substance of interest is required. For this reason, isolating a substance of interest from a mixture of substances is often necessary in many biochemical laboratories. There are many ways to separate substances: on the basis of size by molecular sieve chromatography or SDS-PAGE, on the basis of binding properties by affinity chromatography, or by isoelectric points (the pH at which the substance has no net charge) by isoelectric focusing. Isoelectric focusing is particularly effective for analyzing microheterogeneous protein species or other species which differ slightly in their chemical. content. 
     Isoelectric focusing with an immobilized pH gradient (IPG), makes true isoelectric focusing possible and significantly improves the reproducibility of the spot distribution along the pH gradient axis of 2-D maps. IPG also makes it possible to focus basic proteins in the gel and to obtain 
     Electrophoresis devices are well known in the art. However, attempts to construct an apparatus which successfully analyzes basic proteins (for example those with pH between 8-12) in a simple, user-friendly manner have previously been unsuccessful. Previous “face up” (gel side up) systems required messy preparation and critical setup to effectively load the sample on the gel. Sample cups had to be placed perfectly perpendicular to the gel (despite rotational freedom) and at the perfect height (despite placement flexibility on the vertical axis) using click stops to provide sample contact with the gel yet avoid crushing it. Newer “face down” (gel side down) systems which are easier to load and run, such as that described in co-assigned and application Ser. No. 09/095,002, now issued as U.S. Pat. No. 6,113,736, the contents of which are hereby incorporated by reference as if recited in full herein, cannot successfully separate basic proteins. Therefore, the present invention provides an apparatus which allows for effective analysis of basic proteins in a compact, simple way. 
     OBJECTS AND SUMMARY OF THE INVENTION 
     In view of the foregoing, it is an object of the present invention to provide a sample loading system that is capable of separating basic proteins by isoelectric focusing on an immobilized pH gradient (IPG) in a “face up” system. 
     It is also an object of the present invention to provide a sample loading means that is user-friendly and relatively clean. 
     It is another object of the present invention to simplify sample loading on “face up” gels. 
     It is a further object of the present invention to provide a means for accurate and uncomplicated sample positioning on the vertical axis, thereby providing adequate but not excessive contact of the sample and sample cup with the gel. 
     It is additionally an object of the present invention to create a gel loading system which is adjustable to different length gels and which allows flexibility of sample cup placement. 
     It is another object of the present invention to reduce the volume of mineral oil required to perform isoelectric focusing on a single gel. 
     These and other objects are satisfied by the present invention which is directed to gel loading systems, methods, and associated containers which are configured to successfully load electrophoresis gels with samples of any pH. In particular, a first aspect of the present invention is directed toward a sample loading assembly for electrophoresis gels comprising a gel holder adapted to hold a gel, two electrode carriers and associated electrodes, a sample loading cup adapted to load the sample onto the upper surface of the gel, and a cover, wherein said electrode carriers are configured such that, in ordinary use, the electrodes will be in electrical connection with the top surface of the gel. Specific embodiments include variations on the electrode placement along the gel surface and methods of electrode connection with the electrophoresis apparatus. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a perspective view of a strip holder, electrode, and sample loading cup according to the present invention. 
     FIG. 1A is a bottom and side perspective view of the strip holder according to the present invention. 
     FIG. 2 is an enlarged view of the electrode and electrode carrier configuration according to the present invention. 
     FIG. 2A is an enlarged cutaway perspective view of the electrode carrier and sample loading cup on a strip holder according to the present invention. 
     FIG. 2B is a cutoff side view of different electrode configurations on electrode carriers according to the present invention. 
     FIG. 2C is a cutoff front and cross sectional side view of half of an electrode, primarily illustrating the curved nature of the gel contact surface according to the present invention. 
     FIG. 3 is a perspective view of a sample loading cup according to the present invention. 
     FIG. 3A is a cutoff front view of a sample loading cup on a strip holder according to the present invention. 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The present invention will now be described more fully hereinafter with reference to the accompanying figures, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Like numbers refer to like elements throughout. Layers and regions may be exaggerated for clarity. 
     Referring to the drawings, FIG. 1 illustrates a preferred sample loading assembly  100 . As shown, the assembly  100  includes a gel strip holder  140 , two electrode carriers  120 , a sample loading cup  130 , and a cover  110 . The entire assembly  100  is preferably used in conjunction with an electrophoresis device such as the IPGphor Electrophoresis Unit (Hoefer Pharmacia Biotech Inc., San Francisco, Calif.). The electrophoresis device used in conjunction with the present invention preferably includes power transfer means such as power supply contact pads  190 A,  190 B. 
     As shown in FIG. 1A, the gel strip holder  140  preferably comprises contiguous upstanding side walls  170 A- 170 D arranged in a substantially rectangular shape to form a frame  170  with a longitudinal length “l”. At one end of the frame  170 , the walls  170 A- 170 D are more preferably arranged to form a slight point which visibly distinguishes the end of the gel strip holder  140  which should contact an anodic power supply contact pad  190 A from the more blunt end that should contact a cathodic power supply contact pad  190 B. The frame  170  also has a bottom  170 E which is contiguous to all the upstanding walls  170 A- 170 D such that the walls  170 A- 170 D and floor  170 E of the frame  170  form a liquid-tight container. 
     In one preferred embodiment, the gel strip holder  140  preferably has electrode contact surface pairs  150 ,  151  on the longitudinal sides of the gel strip holder frame  170  (electrode contact surface  150 ,  151  is on both sides of the gel strip holder  140 , only one side shown) as shown in FIG.  1 A. There are preferably two electrode contact surface pairs  150 ,  151  with a non-conductive material between the pairs providing a gap “g” such that the the electrode contact surface pairs  150 ,  151  are not in electrical contact with each other. This enables the gel strip holder  140  to have one pair of electrically isolated (electrically isolated from the other pair) electrode contact surfaces  150 ,  151  for the anode and another for the cathode. Preferably, the gap “g” between the electrode contact surface pairs  150 ,  151 . is no longer than the length of the gel. Additionally, a power contact surface  152 ,  153  on the bottom of the frame  170 E electrically connects the pair of electrode contact surfaces  150 ,  151 . The power contact surfaces  152 ,  153  thereby transfer power from the power supply contact pad  190 A,  190 B on the electrophoresis device to their corresponding pair of electrode contact surfaces  150 ,  151  when the gel strip holder  140  is placed on power supply contact pads  190 A,  190 B. Preferably, the power contact surfaces  152 ,  153  and electrode contact surfaces  150 ,  151  comprise an electrically conductive material which does not corrode or rust when exposed to mineral oil and/or water. Alternatively or additionally, a coating can provide corrosion resistance and/or electrical conductivity. A preferred coating for the electrode contact surfaces  150 ,  151  and power contact surfaces  152 ,  153  is two coats of moly manganese and one coat of nickel-plated oxide. In a preferred embodiment, as shown in FIG. 1A, the power contact surfaces  152 ,  153  extend at least partially lengthwise along the gel strip holder  140 . As is known in the art, the power supply contact pads  190 A,  190 B can be constructed so that the contact pad for the cathode  190 B is small to facilitate positioning of the gel strip holder  140  on the electrophoresis device. The power supply contact pad for the anode  190 A, on the other hand, can advantageously be large to accommodate different length gels as described above. In one preferred embodiment, the power contact surfaces  152 , 153  are sufficiently short as to prevent shorting across the power supply contact pad  190 A,  190 B even if the gel strip holder  140  is placed on the power ,supply contact pad  190 A,  190 B backwards. Preferably, the power contact surface for the anode  152  and cathode  153  are sufficiently large to provide good electrical contact with the power supply contact pad  190 A,  190 B even when the power supply contact pad  190 A,  190 B is not perfectly flat. 
     Furthermore, the gel strip holder frame  170  preferably comprises a material that is nonconductive and provides efficient heat transfer and temperature control. One preferred material is aluminum oxide. The internal sides of the frame  170  are more preferably additionally chemically modified to minimize protein adsorption. Modifications of this type are well known to those of skill in the art, such as a gas phase silane treatment. 
     The holder  140  is furthermore preferably configured to hold a single gel strip  200  of any size, ranging in length from 7 cm to 24 cm. Advantageously, single gel capacity minimizes the volume of mineral oil required to fill the gel strip holder  140 . As shown in FIGS. 1 and 1A, the gel strip holder  140  preferably additionally includes protrusions  180  on the inside of the frame  170 , which contact a gel strip  200  when the gel  200  is in place and additionally keep the gel  200  substantially longitudinally straight. Preferably, these protrusions  180  are substantially small such that they do not inhibit movement of and contact with the sample loading cup  130  and electrode carriers  120  and associated electrodes  220  regardless of gel length as described hereinbelow. As shown in FIG. 1A, the protrusions  180  more preferably comprise substantially rounded geometries at the gel-protrusion interface, so as to not cut or damage the gel  200 . 
     As shown in FIG. 2, in a preferred embodiment, the electrode carriers  120  preferably comprise two legs  272  and a body  271  connecting the two legs  272 . The body  271  is preferably substantially perpendicular to the two legs  272  such that when the electrode carrier  120  is placed on top of the gel strip holder  140 , the electrode carrier  120  wraps around the outside of the gel strip holder  140  as shown in FIG.  2 . The electrode carriers  120  are preferably made of a nonconductive material such as polycarbonate. 
     Each electrode carrier preferably is firmly attached to an electrode  220 . More preferably, the electrodes  220  are comprised of two legs  280  and a body  270  which connects the two legs  280  at substantially right angles as shown in FIG.  2 . Substantially near the center of the electrode body  270 , the electrode  220  preferably projects substantially vertically downward at an angle  251 . After a predetermined distance, the electrode preferably forms a second angle  252  to form a section which is substantially perpendicular to its legs  280 . After a predetermined distance, the electrode  220  preferably projects upward again at a third angle  253  to the inner surface of the electrode carrier  120  and after a fourth angle  254 , projects in substantially the same plane as the initial section of body  270 . The lower section of electrode  220  that is perpendicular to the legs  280  forms a gel contact face  290 . Preferably, all angles  251 - 254  are substantially the same magnitude so that the gel contact surface  290  is substantially parallel to the electrode body  270  as shown in FIG.  2 . The projections therefore preferably form a loop which is substantially flat on the bottom as shown in FIG.  2 . The electrode may be of many different shapes, as shown in FIG. 2B, as long as the gel contact surface  290  is flat. Preferably, the interior angles formed adjacent to the gel contact surface  290  ( 252 ,  253 ) are large enough to inhibit capillary wicking of any substance on or near the gel, and comprise substantially blunt edges to advantageously minimize the possibility of sharp corners damaging the gel as shown in FIG.  2 C. The electrodes preferably comprise an electrically conductive material. More preferably, the electrodes comprise platinum-coated titanium. 
     In operation, the electrode carriers  120  are positioned on the gel strip holder  140  such that one electrode  220  is in electrical contact with the anode of the power supply contact pad  190 A, and the other electrode  220  is in electrical contact with the cathode  190 B of the power supply contact pad. To accommodate different length gels, the electrodes preferably can be placed at various longitudinal locations along the gel strip holder  140  while maintaining electrical contact with the power supply contact pads  190 A,  190 B as described above. Preferably, the electrodes  220  contact the power supply contact pads  190 A,  190 B via the electrode contact surfaces  150 ,  151  and the power contact surfaces  152 ,  153 . 
     In a more preferred embodiment, the electrodes  220  also include at least one contact boss  260  to facilitate firm electrical contact with the electrode contact surfaces  150 ,  151  (FIG.  2 ). More preferably, the electrode  220  includes one contact boss  260  on each side. Additionally, the electrode carriers  120  and electrodes  220  are preferably sized and shaped such that the body of the electrode  270  and electrode carrier  271  act as springs which compress the two legs of the electrode  280  towards each other. This advantageously forces adequate electrical contact between the electrodes  220  and the electrode contact surfaces  150 ,  151 . 
     Advantageously, the present invention allows for the addition of a filter paper wick  210  if necessary to absorb excess water and proteins beyond the pH range of the strip being used. Because the electrode holder  120  (and therefore the electrode  220 ) are not vertically physically supported by the gel strip holder  140  or power supply contact pads  190 A,  190 B, and because the electrode contact surface pairs  150 ,  151  preferably cover substantially the height of the gel strip holder  140 , the electrode  220  within the electrode holder  120  advantageously has continuous height adjustment. Therefore, addition of filter paper wicks  210  of any reasonable height can be accommodated by the present invention. In a preferred embodiment, the force applied to the electrode holder  120  and electrode  220  from the cover  110  cause the electrode  220  to apply the optimal force to the gel strip  200  beneath it. Advantageously, in this embodiment, the electrodes  220  do not damage the gel  200  yet make sufficient contact with the gel  200  regardless of the presence or absence of a paper wick  210 . 
     To provide improved focusing patterns and give better resolution in conditions which entail substantial electroendosmosis, the present invention is designed to run electrophoresis “face up.” Therefore, a means of loading sample from the top is required. A preferred embodiment of the sample loading cup  130  is shown in FIG.  3 . The cup preferably comprises a non-conductive material such as a plastic or a ceramic to allow contact of the cup  130  with the electrode contact surfaces  150 ,  151  without deleterious effects. More preferably, the cup comprises a plastic such as polycarbonate. The sample loading cup  130  is preferably comprised of two legs  320  and a body  310  connecting them. Near approximately the center of the body  310 , there are preferably two longitudinal slanted projections  330 ,  331  which project from the body  310  toward each other. Additionally, there are two substantially perpendicular projections  332 ,  333  which connect the two longitudinal slanted projections  330 ,  331  at their ends as shown in FIG.  3 . These four projections  330 - 333  form an enclosure which acts as a sample chamber  340 . Preferably, the projections  330 - 333  are sufficiently short and spaced apart such that they do not meet at the bottom as shown in FIG.  3 A. Therefore, there is a slot  350  at the bottom of the sample chamber  340  which allows sample within the sample chamber  340  to contact the surface of the gel  200 . Preferably, the slot  350  at the bottom is between 0.5 mm and 2 mm deep, and more preferably the slot is 0.9 mm deep (denoted “d” on FIG.  3 A). Additionally, the sample chamber  130  is preferably the width (denoted “w s ” on FIG. 3) of the gel  200  or less to prevent leakage of sample into the gel strip holder  140  without contacting the gel  200 . 
     More preferably, as shown in FIGS. 3 and 3A, the sample cup  130  further includes at least one friction block  360  on at least one leg  320 . The friction block  360  preferably comprises a small flat sided projection which comprises a suitable contact surface  365  to contact the gel strip holder  140 . The friction block  360  is preferably large enough to coerce contact between the gel strip holder  140  and the friction block  360  when the sample loading cup  130  is placed on the gel strip holder  140  as shown in FIG.  3 A. The friction blocks  360  thereby advantageously keep the sample loading cup  130  at a specific longitudinal location relative to the gel strip holder  140 . In a more preferred embodiment, the sample cup  130  can be placed almost anywhere along the gel strip holder  140  and held in place by at least one friction block  360 . 
     Additionally, the sample loading cup  130  as shown in FIG. 3A also preferably comprises at least one standoff foot  370 , and more preferably two or more. The standoff foot  370  includes two projections  371 ,  372  whose edges are spaced apart a distance “w p ” which is at least the width of a standard-sized rehydrated gel (approximately 3 mm). The standoff foot  370  therefore advantageously centers the gel  200  under the sample chamber  340 . Additionally, the standoff foot  370  is preferably rigidly connected to the sample loading cup  130  and has a sufficiently wide base “b” to provide a stable mounting surface which prevents pivoting motions, thereby advantageously supplying good contact between the gel  200  and the sample loading cup  130  each time the cup  130  is put in place. Furthermore, the standoff foot  370  positions the sample loading cup  130  at the appropriate vertical height “h f ” so that the sample chamber  340  is suitably positioned in relationship to the gel  200  to contact but not crush the gel  200 . This can be accomplished with a single standard size sample loading cup  130  with non-adjustable feet  370  because rehydrated gels  200  are substantially consistent in height (usually around 0.5 mm) regardless of length. A single-sized (i.e. non-adjustable) sample loading cup  130  is therefore advantageous because the operator does not need to adjust and align the sample loading cup  130  on the gel  200 , thereby eliminating the possibility for human error. 
     In a more preferred embodiment, the standoff foot  370  is designed such that when the sample loading cup  130  and electrode carriers  120  are in place, the height “h c ” of the sample loading cup  130  is less than the height “h e ” (see FIG. 2) of the electrode carriers  120 . Therefore, when a cover  110  is placed over the entire assembly as shown in FIG. 1, the cover  110  contacts only the body of the the electrode carriers  270  and not the body of the sample loading cup  310 . This advantageously prevents any sample in the sample chamber  340  from exiting the chamber  340  through the top of the chamber  340  by capillary action. 
     The cover  110  preferably comprises a translucent or transparent non-conductive material. Preferably, the cover  110  is configured to fit loosely over the gel strip holder frame  170 . Advantageously, the cover  110  additionally ensures electrical contact between the gel strip  200  and the electrodes  220  as discussed hereinabove. Furthermore, the cover  110  forces the electrodes  220  to retain their longitudinal location after placement. The use of a cover  110  additionally ensures that the power contact surfaces  152 ,  153  contact the power supply contact pads  190 A,  190 B because it applies pressure to the gel strip holder  140  power supply contact pads  190 A,  190 B interface when the cover of the electrophoresis machine (not shown) applies pressure to it. 
     In operation, the gel loading assembly  100  is assembled as follows. A gel  200  of the desired length is rehydrated face down in a separate container using mineral oil. The rehydrated gel  200  is then placed in the gel strip holder  140  such that the pointed side of the gel  200  is in the pointed side of the gel strip holder  140  and the blunt end of the gel  200  is at the blunt side of the gel strip holder  140 . The gel strip holder  140  and gel  200  are then placed on an electrophoresis machine such that the power contact surfaces  152 ,  153  on the gel strip holder  140  contact the power supply contact pads  190 A,  190 B on the electrophoresis machine as shown in FIG.  1 . An amount of mineral oil is then applied to the gel  200  in the gel strip holder  140  to submerge the gel  200  with mineral oil (generally between 2 to 10 mLs). At this point, the electrode carriers  120  are placed such that they straddle the gel strip holder  140  as shown in FIG.  2 . The sample loading cup  130  is then similarly placed in the gel strip holder  140 , making sure that the standoff feet  370  straddle the gel  200  and that the friction blocks  360  are in firm contact with the gel strip holder  140  as shown in FIG.  3 . Finally, sample (up to 100 μL) is placed in the sample chamber  340  and the cover  110  is placed over the gel strip holder  140 . If desired, a blotted wet paper wick  210  can be placed on the gel  200  (where the electrode  220  will be placed such that it ends up between the electrode  220  and the gel  200 ) before the electrode  220  is put in place. 
     It is apparent that many modifications and variations of the invention as hereinabove set forth may be made without departing from the spirit and scope thereof. The specific embodiments described are given by way of example only, and the invention is limited only by the terms of the appended claims.