WO1990013020A1

WO1990013020A1 - Non-glass gel mold having low oxygen permeability and uses thereof

Info

Publication number: WO1990013020A1
Application number: PCT/US1990/002213
Authority: WO
Inventors: Sheldon C. Engelhorn; Richard T. L. Chan
Original assignee: Novel Experimental Technology
Priority date: 1989-04-25
Filing date: 1990-04-24
Publication date: 1990-11-01

Abstract

A non-glass gel mold comprising a material having an oxygen permeability coefficient under gel forming conditions sufficient to reduce or prevent band smearing in a separation technique is provided. A method of making a gel utilizing the gel mold and the gel made therefrom are also provided. Additionally, a method of reducing or preventing smearing in a gel during a separation technique comprising performing the separation in the formed gel is also provided.

Description

1

NOVEL-_GLASS GEL MOLD HAVING LOW OXYGEN PERMEABILITY AND USES THEREOF

BACKGROUND OF THE INVENTION

The invention relates generally to gel molds useful in separation techniques. Specifically, a non-glass gel mold having low oxygen permeability is provided.

Gel electrophoresis is a well-known technique used to separate large molecules or aggregates. A number of related techniques, for example, Isoelectric focussing and western blotting, are also commonly utilized . These techniques can be used to assess the purity of such materials. Typically, gel electrophoresis is used in biological research where the materials separated are proteins, glycoproteins, polypeptides, nucleic acids (DNA, RNA) , and other biological materials.

Gels are generally prepared by pouring a liquid mixture into a container or mold, and allowing polymerization to take place. A typical liquid -mixture consists of acrylamide, water, bisacrylamide, and a free-radical-producing initiator. The acrylamide polymerizes to a lightly cross-linked gel which holds its size and shape in a manner similar to gelatin and other such materials.

The acrylamide gel is a porous, water-filled structure through which molecules below a certain size can move by diffusion or under the influence of an electric field. Application of an electric field to a gel containing the large molecules or aggregates results in movement of charged molecules through the gel with a speed (or mobility) according to their charge and size. Since a variation in the transport properties of the gel (porosity, pore size, frequency of cross-links, etc.) will result in a different speed of travel for the materials being separated or tested for purity, uniformity of the gel structure is important. Gels with various transport properties (pore size, porosity, etc.) are produced by controlling the polymerization conditions and result in different gels suitable for the 2 separation of molecules of varying sizes.

Molds for gels are typically glass tubes or plates (with spacing rails between the plates to provide space for the formation of a thin slab of gel) with the ends accessible for introducing samples, buffer solutions, and applying the electric field. A small sample is applied to an indentation (or "well") in the gel at one end, and an electric field is applied for periods of time from minutes to hours. Under conditions typical for such separations, a pure sample ideally will form a single, sharp, well-defined band resulting from uniform migration of the molecules or aggregates. Impurities that move at another speed will be spread out into a series of bands that may merge into a smear. Since the primary purpose of gel electrophoresis is to separate and identify materials based on their size and charge, variations in gel characteristics prevent uniform migration and therefore are highly undesirable. If one portion of the sample is moving in an area of gel with different pore characteristics than the rest of the sample, the bands will not i>e well defined and a pure sample will appear heterogeneous.

Glass gel molds have the advantage of generally molding gels which have uniform characteristics and give clear, sharp bands after electrophoresis. However, glass has disadvantages in that it is breakable, expensive, difficult to shape and is non-disposable. As a result of the disadvantages associated with glass, plastic gel molds which may be small, inexpensive and disposable have increased greatly in use. Because of these characteristics, plastic molds are especially useful in purity determinations.

While having many advantages, currently used plastic molds leave a band smear which is highly undesirable in a separation technique. Since this smear is apparent even when the sample is highly purified, currently utilized plastic molds are much less desirable in purification analysis. Thus, there exists a long felt need for a non-glass gel mold having the advantages of plastic but which reduce or prevent band smearing in a separation technique. This need is satisfied and other related advantages are provided by the subject invention.

SUMMARY OF THE INVENTION

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the absence of smearing in a gel formed with a glass mold.

Figure 2 shows the smearing effect of a gel formed with an acrylic plastic mold.

Figure 3 shows the smearing effect of a gel formed by a glass mold coated with low density polyethylene.

Figure 4 shows the smearing effect of a gel formed by a glass mold coated with polyester film.

Figure 5 shows the smearing effect of a gel formed by glass plates with a sheet of polyester film covering one of the plates.

Figure 6 shows the absence of smearing in a gel formed with acrylic plates coated with a latex consisting of a vinylidene chloride-acrylate copoly er dispersion in water. Figure 7 shows the smearing effect of a gel formed with glass covered with a layer of polyvinyl chloride (PVC) film.

Figure 8 shows the absence of smearing in a gel formed with glass covered with a layer of polyvinylidene chloride (PVPDC) coated PVC film.

Figure 9 shows the absence of smearing in a gel formed with acrylic plates covered with a layer of poly(acrylonitrile- co-methyl aerylate) .

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a non-glass gel mold comprising an amount of a material having an oxygen permeability coe ficient under gel forming conditions sufficient to reduce or prevent band smearing in a separation technique. Since it is known that oxygen inhibits free radical pol^*ymerization of acrylamide and other materials polymerized by similar reactions, the gel forming solution is typically kept from direct contact with air. In addition, the solution is deoxygenated prior to initiation of polymerization or sufficient catalyst is added to overcome the inhibition of dissolved oxygen. It has now been discovered that sufficient oxygen can pass from the gel mold into the gel as it polymerizes to cause partial inhibition of polymerization and alter polymer porosity along the surface of the mold. Prior to this discovery it had not been recognized that typical gel mold materials, such as poly (methyl methacrylate) or polyterepthalate can release oxygen and form a chemically significant oxygen gradient in the polymerizing solution perpendicular to the wall-gel interface. While this is not the case for glass-walled molds, many commonly used plastics, such as polystyrene, polycarbonate, and polyethylene, have relatively high solubility for oxygen and high permeability for oxygen relative to glass. Gel porosity is altered at the interface even though polymerization proceeds to 5 completion with no visually apparent difference between gels polymerized in such plastic molds and gels polymerized in glass molds. The mold material is generally in equilibrium with the atmosphere before use, and even after some time in an oxygen-free environment most plastic materials slowly release oxygen for extended periods of time. Alternatively, the mold material may absorb oxygen from the gel mixture and the oxygen so absorbed can diffuse back into the gel during polymerization. The permeability levels required for this mechanism are not permeation of atmospheric oxygen from the outside of the mold walls, but are from a region in the wall material close to the gel-wall interface, to that interface. This permeation probably involves total distances of less than a few tenths of a millimeter, depending on the wall material used.

It has also been discovered that this lack of polymerization along the surface of gel causes large molecules to migrate faster in such areas during electrophoresis. It has still further been discovered that this lack of polymerization and the associated variance in migration -rate of molecules of the same size and charge causes the bands to smear during electrophoresis. Since inhibition of polymerization causes the affected portion of the gel to develop a more porous structure, the smear results from the sample moving more rapidly in regions of the gel near the gel-wall interface. When the sample is subsequently visualized, the effect is manifested as a smear in front of the sharp band that is found in the bulk of the gel (away from the interface) . This effect is particularly deleterious for a purity assessment, as it mimics the pattern that would be expected for a sample with impurities, particularly degraded samples.

Additionally, it has now been discovered that a non-glass gel mold comprising a sufficient amount of a material of sufficiently low oxygen permeability, as expressed in the oxygen permeability coefficient, is effective in reducing or preventing band smearing. By "reducing" is meant any reduction in band smearing over the presently employed non- 13020

6 glass molds, for example, polystyrene, polycarbonate or polyethylene.

Generally, materials having permeability coefficients under gel forming conditions of less than .04 X 10^"10 units are effective in reducing band smearing. However, as a general rule, the lower the oxygen permeability coefficient the better the gel polymerization adjacent to the gel and the sharper the bands resulting from a separation technique. Thus, a permeability coefficient of less than .02 X 10^"10 units is preferred over .04 X 10^*10 units and .01 X 10^*10 units is preferred over .02 X 10^*10 units. Additionally, applicants have found that oxygen permeability coefficients of about .005 X 10^*10 units or less result in sharp bands with no, or very minimal, smearing and thus is preferred. The units referred to in the permeability coefficient are

(CM³ of 0₂ at STP) X (CM thickness)/[(CM² surface area) X sec X CM Hg (pressure across surface) ] (hereinafter expressed merely as "units") .

It will be recognized that if materials of low oxygen permeability are mixed with materials having a higher oxygen permeability, a proportionally larger amount of the lower permeability material will be required to obtain an effective mixture. Moreover, in mixtures of materials used in a gel mold, the overall oxygen permeability must be sufficiently low to prevent or reduce band smearing, i.e., less than .04

X 10^"10 units, preferably .02 X 10^"10 units, more preferably

.005 X 10^"10 units. For example, if a material having an oxygen permeability of 0.1 X lθ^"10 units is mixed equally with a material having an oxygen permeability coefficient of .04 X 10^"10 units, the mixture would not be effective in preventing or reducing band smearing. However, if the lower permeability material is coated on the higher permeability material so as to interface the gel, it may be effective.

The oxygen permeability coefficients of various materials are either known or readily measurable. However, some materials do not retain a sufficiently low oxygen 7 permeability coefficient under "gel forming conditions." By "under gel forming conditions" is meant the environment prior to and during polymerization. For example, a material should maintain a sufficiently low oxygen permeability in the aqueous environment created by the non-polymerized gel to allow adequate polymerization adjacent to the gel mold material. The effect of an aqueous environment can be readily determined by placing the material in the aqueous environment.

The material of this invention can be used in a coating separating ^*the mold from the gel. Alternatively, the mold itself can be made of the material or an effective mixture of the material. The material is preferably immediately adjacent to the gel and of sufficient thickness to prevent or reduce oxygen entering the polymerizing gel. Examples of suitable materials are polymers, preferably polyvinylidine chloride or poly(acrylonitrile-co-methyl acrylate) . Another example of the material is vinylidene chloride-acrylate. Moreover, the material can be used in any mixture which is effective and lias a sufficiently low oxygen permeability coefficient.

The separation technique utilizing the provided gel mold can be any separation technique in which gel polymerization is required. One common example of a separation technique is electrophoresis.

A method of making a gel which reduces or prevents band smearing in a separation technique and the gel formed therefrom is also provided. The method comprises forming the gel in the gel mold described. The separation technique can be electrophoresis. The gel can be polyacrylamide or any solution which can be polymerized and used in a separation technique.

The invention also provides a method of reducing or preventing smearing in a gel during a separation technique comprising performing the separation in the gel described. 13020

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Further, a method of determining the purity of a sample comprising performing electrophoresis in the gel of the invention and observing the sample bands is provided. In this method non-smeared bands indicate a pure sample.

Finally, a method of separating components in a sample is provided. The method comprises loading samples onto the described gel, subjecting the gel to an electric current sufficient to cause migration of the components, terminating the electric current, detecting the separated components on the gel. The method comprises the known features of gel electrophoresis but is carried out in a gel made by a non- glass gel mold which reduces or prevents band smearing. Thus, the method provides clear gel bands.

The following examples are given for the purpose of illustration and are not intended to limit the invention contained herein.

EXAMPLE I

The following mixture was prepared and degassed by nitrogen purge for 30 seconds prior to pouring:

Acrylamide 12 g

Bis-acryiamide 0.32 g

1.875 molar Tris/HCl, pH 8.9 20 ml

TEMED 125 μl

Ammonium Persulfate (10%) 400 μl (added before pouring) Water to bring final volume to 100 ml

About 5 ml of this solution was poured between two flat glass plates about 10 cm square separated by about 0.6 mm from each other with plastic spacers and sealed around 3 sides to contain the solution. The inner dimensions of the mold were about 8cm X 8cm X 0.6mm. This solution filled the mold to a height of about 6 cm. Water was carefully layered over the gel solution and polymerization was allowed to proceed. Gel formation occurred in about 10 minutes. After 9 the solution gelled the water was removed and a solution (stacking gel) prepared as above but with 60% lower acrylamide and Bis-acrylamide was added to completely fill the mold. A well forming "comb" was inserted between the plates and the second solution was allowed to polymerize. After several hours to allow complete polymerization, the gels were ready to be used.

In one well 5 μl, and in an adjacent well, 15 μl of a protein mixture consisting of 0.1 mg/ml each of Rabbit phosphorylase b (97,400 Daltons) , Bovine Serum Albumin (66,200 Daltons), Hen Ovalbumin (42,700 Daltons), Soybean Trypsin inhibitor (21,500 Daltons) , Hen egg white lysozyme (14,400 Daltons). In two adjacent wells (5 μl and for one and 15 μl for the other) lysozyme alone at a concentration of about 1 mg/ml was applied. All proteins were dissolved by heating briefly in a solution containing about 0.125 molar Tris/HCl pH 6.8, 10% glycerol, 2% Sodium dodecyl sulfate, 5% 2-mercaptoethanol, and 0.05% Bromophenol Blue. The buffer for the electrode tanks was 24 M Tris, 192 ^*mM Glycine, 0.1% SDS. The gels were run at about 125 volts for about 1.5 hours with the proteins migrating toward the anode.

Upon completion of the run, the gel was removed from the mold and placed in a solution of 0.1% Coomassie Blue R-250, 40% methanol, 10% acetic acid, for about 20 minutes with gentle agitation to stain the proteins. The background stain was removed by placing the gel in a solution of 10% methanol, 7.5% acetic acid with gentle agitation for 1 to 3 hours. The destaining solution was changed several times during this interval.

The gel is shown with the direction of migration from top to bottom. The proteins separate in order of size with the smallest, lysozyme, at the bottom. The bands are distinct with the leading edge of the bands particularly well defined as is typical of results using this method. 13020

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EXAMPLE II

A solution prepared in the manner of Example I was introduced into a mold as before but with the mold surfaces consisting of molded acrylic plastic (poly(methyl methacrylate) ) .

As seen in Figure 2, the same protein bands are observed as before but the bands are less distinct and a faint smear is evident extending before each band. The band intensity of the lighter bands is lower than for the previous example as ^•the smear consists of material which in the previous example is confined to the main bands. The acrylic plastic typically has a permeability coefficient for oxygen of about

0.1 X 10^"10 (CM³ of 0₂ at STP) X (CM thickness)/[ (CM² surface area) X sec X CM Hg (pressure across surface) ] . (Data provided by Carolyn Sary, Plastics Technology Center, Rohm & Hass Corporation, Bristol, PA)

EXAMPLE III

A solution was prepared and introduced into a mold in the manner of Example I but with the gel contact surfaces of the mold coated on the gel contact side with a film about 0.2mm thick of low density polyethylene.

As can be seen in Figure 3, the band definition and intensity is further reduced as compared to Example II and the smearing effect is increased. Low density polyethylene typically has a permeability for oxygen of about 3.0 X 10^"10 units (Polymer Handbook, 2nd edition, Brandrup & Immergut, Published by John Wiley, N.Y., 1975).

EXAMPLE IV

A solution was prepared and introduced into a mold in the manner of Example I but with the gel contact surfaces of the mold coated with polyester film (polyterephtalate) of about 11

0.2mm thickness. The bands are more distinct than for Example II or III. As seen in Figure 4, the smearing effect is also evident but not as severe as in Examples II and III, in that the length of the smear is shorter. The polyester typically has a permeability for oxygen of about 0.04 X 10 ¹⁰ units (Polymer Handbook, supra) .

EXAMPLE V

A solution was prepared and introduced into a mold in the manner of Example I but with a 0.2mm sheet of polyester film (of the same plastic composition and permeability as in Example IV but with one surface prepared according to the United States Patent No. 4,415,428, which is incorporated herein by reference) covering one of the plates and with the treated surface of the film in contact with the gel solution. For this Example, the gel remains attached to the film for the remainder of the processing. As seen in Figure 5, the bands are similar to those for Example IV. For Example V the dimensions were changed to 8cm X 8cm X 0.3mm to compensate for the fact that only one side of the gel is in contact with the plastic surface. The other side is in contact with glass.

EXAMPLE VI

A solution prepared in the manner of Example I was introduced into a mold in the manner of Example II but with the surface of the acrylic plates coated with a layer of latex consisting of a Vinylidene Chloride-Acrylate copolymer dispersion in water. The cured thickness of the layer is about 0.03mm. Such a material, supplied for providing oxygen and moisture barrier layer, is available under the trade name Daran 8680 (W.R. Grace, Lexington, MA) Immediately after brushing on a thin layer of the latex the surface was dried for about 30 seconds by radiant heat. The plates were then assembled as before. As seen in Figure 6, little or no difference in band intensity or morphology as compared to /13020

12

Example I could be observed. Little or no smearing is observed. The permeability of the coating for oxygen is less than .001 X 10 ^"10 units (data from Daran 8680 Technical Data Sheet, W. R. Grace, Lexington, MA) .

EXAMPLE VII

A solution was prepared and introduced into a mold in the manner of Example I but with the gel contact surfaces of the mold covered with a 0.2 mm layer of polyvinyl chloride film. As seen in Figure 7, relative to glass, the effect on band morphology and smearing was intermediate between that of Examples I and II. The oxygen permeability of the PVC film is about 0.05 X 10 ^{" 0} units (Polymer Handbook, supra) .

EXAMPLE VIII

A solution was prepared and introduced into a mold in the manner of Example I but with the gel contact surfaces of the mold ccvered with a 0.05 mm layer of polyvinylidene chloride (PVDC) coated PVC film (0.2 mm) with the polyvinylidene coated surface in contact with the gel solution. As seen in Figure 8, little or no difference in band intensity or morphology as compared with Example I is observed. Little or no band smearing is observed. PVDC has a permeability to oxygen of less than 0.001 X 10 ^"10 units (data provided by Knud Christiansen, Klockner-Pentaplast Inc. , Gordonsville, VA) .

EXAMPLE IX

A solution was prepared and introduced into a mold in the manner of Example I but with the gel contact surfaces of the mold covered with a 0.02 mm layer of Poly(acrylonitrile-co- methyl acrylate-co-nitrile rubber) (Barex 210™, British Petroleum, OH) . As seen in Figure 9, little or no difference in band morphology or intensity is observed between this Example or Example I. Little or no smearing is observed. 13

Permeability of Poly(acrylonitrile-co-methyl acrylate-co- nitrile rubber) for oxygen is about 0.005 X 10 ^'10 units (Data from British Petroleum brochure #B210-02) .

It will be understood that other materials with similar barrier properties may be used in place of those given in the examples, including multiple layers of materials such as adhesives, binders, and the like. Suitable barriers may include metal foil if removed from the solidified gel before electrophoresis, or if electrically insulated from the gel by a thin layer of nonconductive material.

While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to those skilled in the art that various changes can be made without departing from the spirit and scope of the invention.

Claims

114What is claimed is:

1. A non-glass gel mold comprising an amount of a material having an oxygen permeability coefficient under gel forming conditions sufficient to reduce or prevent band smearing in a separation technique.

2. The mold of claim 1, wherein the material has a permeability coefficient under gel forming conditions of less than about .04 X 10^"10 units.

3. The mold of claim 1, wherein the material has a permeability coefficient under gel forming conditions of less than about .02 X 10^"10 units.

4. The mold of claim 1, wherein the material has a permeability coefficient under gel forming conditions of less than about .005 X 10^"10 units.

5. The mold of claim 1, wherein the material is adjacent to the gel.

6. The mold of claim 1, wherein the material is a polymer.

7. The mold of claim 6, wherein the polymer is selected from the group consisting of polyvinylidene chloride andpoly(acrylonitrile-co-methylacrylate-co-nitrilerubber) .

8. The mold of claim 1, wherein the material is vinylidene chloride-acrylate.

9. The mold of claim 1, wherein the separation technique is electrophoresis. 15

10. The mold of claim 1, wherein the material is a coating adjacent to the gel.

11. A non-glass gel mold having an oxygen permeability coefficient under gel forming conditions of less than the permeability coefficient of polyester.

12. A non-glass gel mold having an oxygen permeability coefficient under gel forming conditions of equivalent to or less than the permeability coefficient of polyvinylidene chloride.

13. A method of making a gel which reduces or prevents band smearing in a separation technique comprising forming the gel in the mold of claim 1.

14. The method of claim 13, wherein the separation technique is electrophoresis.

15. The -method of claim 13, wherein the gel is polyacrylamide.

16. A gel formed by the method of claim 13.

17. A method of reducing or preventing smearing in a gel during a separation technique comprising performing the separation in the gel of claim 16.

18. A method of determining the purity of a sample comprising performing electrophoresis in the gel of claim 16, observing the sample bands, non-smeared bands indicating a pure sample.

19. A method of separating components in a sample comprising:

(1) loading samples onto the gel of claim 16;

(2) subjecting the gel to an electric current i sufficient to cause migration of the components; 13020

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(3) terminating the electric current;

(4) detecting the separated components on the gel.

20. In a method of separating components in a sample by gel electrophoresis, the improvement comprising performing the separation in the gel of claim 16.