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WO1990013020A1 - Moule non vitreux pour gels a faible permeabilite a l'oxygene et ses utilisations - Google Patents

Moule non vitreux pour gels a faible permeabilite a l'oxygene et ses utilisations Download PDF

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Publication number
WO1990013020A1
WO1990013020A1 PCT/US1990/002213 US9002213W WO9013020A1 WO 1990013020 A1 WO1990013020 A1 WO 1990013020A1 US 9002213 W US9002213 W US 9002213W WO 9013020 A1 WO9013020 A1 WO 9013020A1
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WO
WIPO (PCT)
Prior art keywords
gel
mold
permeability coefficient
smearing
glass
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1990/002213
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English (en)
Inventor
Sheldon C. Engelhorn
Richard T. L. Chan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novel Experimental Technology Inc
Original Assignee
Novel Experimental Technology Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novel Experimental Technology Inc filed Critical Novel Experimental Technology Inc
Publication of WO1990013020A1 publication Critical patent/WO1990013020A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories

Definitions

  • 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.
  • 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.
  • Glass gel molds have the advantage of generally molding gels which have uniform characteristics and give clear, sharp bands after electrophoresis.
  • glass has disadvantages in that it is breakable, expensive, difficult to shape and is non-disposable.
  • 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.
  • 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.
  • 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.
  • PVC polyvinyl chloride
  • Figure 8 shows the absence of smearing in a gel formed with glass covered with a layer of polyvinylidene chloride (PVPDC) coated PVC film.
  • PVPDC polyvinylidene chloride
  • Figure 9 shows the absence of smearing in a gel formed with acrylic plates covered with a layer of poly(acrylonitrile- co-methyl aerylate) .
  • 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.
  • 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.
  • 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.
  • 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.
  • reducing is meant any reduction in band smearing over the presently employed non- 13020
  • glass molds for example, polystyrene, polycarbonate or polyethylene.
  • materials having permeability coefficients under gel forming conditions of less than .04 X 10 "10 units are effective in reducing band smearing.
  • 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.
  • 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
  • CM 3 of 0 2 at STP X (CM thickness)/[(CM 2 surface area) X sec X CM Hg (pressure across surface) ] (hereinafter expressed merely as "units”) .
  • 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.
  • 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.
  • suitable materials are polymers, preferably polyvinylidine chloride or poly(acrylonitrile-co-methyl acrylate) .
  • Another example of the material is vinylidene chloride-acrylate.
  • 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
  • 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.
  • non-smeared bands indicate a pure sample.
  • a method of separating components in a sample 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.
  • 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).
  • 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
  • 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
  • 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) ) .
  • 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
  • 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.
  • 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).
  • the polyester typically has a permeability for oxygen of about 0.04 X 10 10 units (Polymer Handbook, supra) .
  • Example II 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.
  • the gel remains attached to the film for the remainder of the processing.
  • the bands are similar to those for Example IV.
  • 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 II 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.
  • 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)
  • Daran 8680 W.R. Grace, Lexington, MA
  • 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) .
  • 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) .
  • PVDC polyvinylidene chloride
  • 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) .
  • 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.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Electrochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Separation Using Semi-Permeable Membranes (AREA)

Abstract

Un moule non vitreux pour gels est composé d'un matériau ayant un coefficient de perméablité à l'oxygène, dans des conditions de formation du gel, suffisant pour réduire ou empêcher la formation de traînées lors de l'utilisation du moule pour des procédés de séparation. L'invention concerne également un procédé de fabrication du gel au moyen de ce moule et le gel ainsi obtenu. Elle concerne en outre un procédé pour réduire ou empêcher la formation de traînées dans un gel lors d'un procédé de séparation où la séparation est effectuée dans le gel déjà formé.
PCT/US1990/002213 1989-04-25 1990-04-24 Moule non vitreux pour gels a faible permeabilite a l'oxygene et ses utilisations Ceased WO1990013020A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US34389189A 1989-04-25 1989-04-25
US343,891 1989-04-25

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WO1990013020A1 true WO1990013020A1 (fr) 1990-11-01

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995031717A1 (fr) * 1994-05-13 1995-11-23 Novel Experimental Technology Moule en matiere plastique pourvu d'un revetement pour gel a electrophorese
US5753095A (en) * 1994-09-19 1998-05-19 Novel Experimental Technology Plastic mold for electrophoresis gel
WO2006057601A1 (fr) * 2004-11-26 2006-06-01 Ge Healthcare Bio-Sciences Ab Composite a base de gel
WO2014081874A1 (fr) * 2012-11-20 2014-05-30 Bio-Rad Laboratories, Inc. Gels d'électrophorèse de polyacrylamide avec protection contre une exposition à l'oxygène

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4897306A (en) * 1986-04-19 1990-01-30 Fuji Photo Film Co., Ltd. Medium for electrophoresis

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4897306A (en) * 1986-04-19 1990-01-30 Fuji Photo Film Co., Ltd. Medium for electrophoresis

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995031717A1 (fr) * 1994-05-13 1995-11-23 Novel Experimental Technology Moule en matiere plastique pourvu d'un revetement pour gel a electrophorese
US5685967A (en) * 1994-05-13 1997-11-11 Novel Experimental Technology Coated plastic mold for electrophoresis gel
US5753095A (en) * 1994-09-19 1998-05-19 Novel Experimental Technology Plastic mold for electrophoresis gel
WO2006057601A1 (fr) * 2004-11-26 2006-06-01 Ge Healthcare Bio-Sciences Ab Composite a base de gel
WO2014081874A1 (fr) * 2012-11-20 2014-05-30 Bio-Rad Laboratories, Inc. Gels d'électrophorèse de polyacrylamide avec protection contre une exposition à l'oxygène
CN104813161A (zh) * 2012-11-20 2015-07-29 生物辐射实验室股份有限公司 保护免于接触氧的聚丙烯酰胺电泳凝胶
US9664646B2 (en) 2012-11-20 2017-05-30 Bio-Rad Laboratories, Inc. Polyacrylamide electrophoresis gels with protection against oxygen exposure

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