WO1997019989A1 - Materiaux a base de polyacrylamide pouvant etre reconstitues et procedes de production - Google Patents

Materiaux a base de polyacrylamide pouvant etre reconstitues et procedes de production Download PDF

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Publication number
WO1997019989A1
WO1997019989A1 PCT/US1995/015289 US9515289W WO9719989A1 WO 1997019989 A1 WO1997019989 A1 WO 1997019989A1 US 9515289 W US9515289 W US 9515289W WO 9719989 A1 WO9719989 A1 WO 9719989A1
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Prior art keywords
gel
polyacrylamide
dehydrated
reconstitutable
solution
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Application number
PCT/US1995/015289
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English (en)
Inventor
Ta-Yun Fang
Original Assignee
Fang Ta Yun
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.)
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Publication date
Application filed by Fang Ta Yun filed Critical Fang Ta Yun
Priority to PCT/US1995/015289 priority Critical patent/WO1997019989A1/fr
Publication of WO1997019989A1 publication Critical patent/WO1997019989A1/fr

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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
    • G01N27/44747—Composition of gel or of carrier mixture

Definitions

  • This invention relates to polyacrylamide gels and methods for producing same for electrophoretic analysis, and more particularly to reconstitutable polyacrylamide materials which can be readily stored and then subsequently formed into polyacrylamide gel samples, as required, for use in electrophoretic analysis .
  • PAGE Polyacrylamide gel electrophoresis
  • Electrophoretic separation of biological molecules on polyacrylamide gel is based on the net charge, shape and size of these molecules. These gels are typically formed by the polymerization of an acrylamide monomer solution. The process is initiated by adding a free radical-generating compound into the acrylamide solution. Gels are made with different concentration of acrylamide and cross linked by adding initiator (such as ammonium persulfate) and an accelerator, for example, tetramethyl-ethylenediamine (TEMED) . A detergent, such as sodium dodecyl sulfate (SDS), or urea are incorporated into the gel if samples are analyzed under dissociation conditions.
  • initiator such as ammonium persulfate
  • accelerator for example, tetramethyl-ethylenediamine (TEMED)
  • a detergent such as sodium dodecyl sulfate (SDS), or urea are incorporated into the gel if samples are analyzed under dissociation conditions.
  • SDS sodium dodecyl sulfate
  • proteins are reacted with the anionic detergent SDS, or sodium lauryl sulfate, to form negatively charged complexes.
  • SDS anionic detergent
  • the amount of SDS bound by a protein, and so the charge on the complex, is roughly proportional to its size. Commonly, about 1.4 g. SDS is bound per 1 g. protein, although there are exceptions to this rule.
  • the proteins are generally denatured and solubilized by their binding of SDS, and the complex forms a prolate ellipsoid or rod of a length roughly proportionate to the protein's molecular weight.
  • proteins of either acidic or basic form negatively charged complexes that can be separated on the bases of difference in sizes by electrophoresis through a sieve ⁇ like matrix of polyacrylamide gel.
  • SDS-polyacrylamide gel electrophoresis is reproducible, versatile, and convenient. It uses either a tubular disc gel or a slab gel.
  • the slab gel technique is an improvement of the disc gel electrophoresis. Less gel and sample materials are required per assay on a slab gel system, and the resulting gel can be easily used for further analysis.
  • Electrophoresis on slab gel of full size (-16-20 x 16 cm) , or mini-size (-10 x 10 cm) of 1.5 m, 1.0 mm, 0.75 mm and even 0.5 mm thickness, is made with composition described by Davis (1964) and by Laemmli.
  • the gel is typically prepared at an acrylamide concentration of 15%, 12%, 10%, 8% or 7.5% or of a gradient in concentration ranging from 5% to 20%. Often, a 4% stacking gel is casted on the top portion of the gel to facilitate good separations.
  • the conventional method to make slab polyacrylamide gel is by casting a polymeric gel between a pair of glass plates.
  • the initiator- containing acrylamide gel solution is cast in the rectangular cavity formed between the two glass plates.
  • the thickness of the gel is determined by the thickness of spacers inserted between the glass plates which form the cavity therebetween.
  • the acrylamide gel solution with TEMED added is poured into the space between glass plates.
  • the gel forms after the acrylamide polymerization is completed.
  • the bottom spacer portion is then removed from the assembly.
  • To carry out electrophoresis the lower part of the gel sits in a buffer bath and the top part of the gel is enclosed by a top buffer bath. Then, electrical current is applied to the system to separate molecules of protein or nucleic acid.
  • the chemical composition of the gel varies depending on specificity of experiments. Poly ⁇ acrylamide gel electrophoresis is divided into denatured, partially denatured and non-denatured PAGE categories . Denatured PAGE system is used for protein and nucleic acid studies. For protein molecular weight determinations, the gel and the electrophoresis buffer include a detergent (SDS) . In this way, the intra-molecular disulfide bonds of sample proteins are reduced before electrophoresis. In protein chemistry, PAGE in the presence of detergent SDS separates mixture of protein based on the molecular weight . During separation, while applying electrical current, diffusion through gel is minimized. Molecules resolve into narrow bands depend on their mobility in the gel matrix. Protein bands are visualized after staining. The gel is dehydrated for preservation or to continue with other analysis .
  • SDS detergent
  • the denatured PAGE system is also used in nucleic acid and gene sequencing studies.
  • the sequencing gel contains urea and/or foramide. After electrophoresis separation, radioactive labeled nucleotides are examined under UV light. The gel is also exposed to X-ray film for establishing a permanent record.
  • a partially denatured PAGE system uses a detergent-containing gel and buffer to assay non- denatured, native proteins.
  • a detergent-containing gel and buffer to assay non- denatured, native proteins.
  • neither gel nor buffer contains detergent. This is used to assay native proteins.
  • Pre-mixed acrylamide powders do not eliminate the potential danger of handling bio-hazardous chemicals .
  • Premixed acrylamide solutions at specific gel strength limit the application of electrophoresis and do not completely eliminate the materials handling problem. Limited shelf life of premixed solution means unavoidable wastes. In any case, the 10% ammonium persulfate solution has to be made the day of casting the gel.
  • Pre-cast mini-gel provides convenience for electrophoresis at high cost with a short shelf life and requirement of special cold storage.
  • the method and system of the present invention overcomes all of the existing needs described above and provides for the formation of a polyacrylamide gel sample with desirable and advantageous properties employing a process which provides the requisite levels of simplicity, reduced cost, and fewer processing steps .
  • a dehydrated reconstitutable material can be prepared in quantities and stored for later use.
  • a dehydrated reconstitutable material can be regenerated in a buffer system to form a regenerated gel .
  • the gel is ready for electrophoresis in about 25 minutes.
  • the polyacrylamide material can be readily regenerated. 5.
  • the regenerated gel of this invention is similar when employed in testing as a freshly made gel and produces comparable electrophoresis resolution of macromolecules.
  • the regenerated gel can be further processed or treated as a freshly prepared gel.
  • the subject method eliminates all the dealing with potentially bio-hazardous chemicals.
  • a dehydrated reconstitutable polyacrylamide material for use as a polyacrylamide gel sample in electrophoretic analysis comprises a polyacrylamide and a stabilizer material, but excludes a buffering salt.
  • the total solids of the dehydrated reconstitutable polyacrylamide material can be from about 5 up to about 40 weight %, preferably 10 up to about 35 weight %, more preferably 12 up to about 32 weight %, and most preferably from about 15 up to about 30 weight %, based on the total weight of the dehydrated reconstitutable polyacrylamide material.
  • the stabilizer material preferably comprises glycerol or sucrose.
  • the amount of said stabilizer material preferably comprises from about 1% up to about 40%, and more preferably from about 2% up to about 30% by weight, and most preferably from about 5% up to about 20% by weight, based on the weight of the acrylamide gel solution.
  • the gel regenerating solution can also include a buffer salt, with or without a denaturing agent. It can also include a detergent and/or a denaturing agent.
  • the method of the present invention can include the further step of storing the dehydrated reconstitutable polyacrylamide material at ambient temperature for future use. This overcomes the need for cold storage.
  • the subject method can include the further steps of providing a gel regenerating solution for regenerating the dehydrated reconstitutable polyacrylamide material, introducing the dehydrated reconstitutable polyacrylamide material into the gel regenerating solution; and regenerating the polyacrylamide gel sample for use in electrophoretic analysis.
  • the method of this invention can include the further steps of, after forming a gel regenerating solution for regenerating the dehydrated reconstitutable polyacrylamide material, providing a gel formation system, introducing the dehydrated reconstitutable polyacrylamide material into the interior of the gel formation system, introducing the gel formation system and the dehydrated reconstitutable polyacrylamide material into the gel regenerating solution, and regenerating the dehydrated reconstitutable polyacrylamide material for use in electrophoretic analysis.
  • This invention relates to a method and system for producing a dehydrated reconstitutable polyacrylamide material which can be formed into a polyacrylamide gel sample typically for use in electrophoretic analysis.
  • a plurality of chemical materials as previously described are employed for producing a polyacrylamide gel used to make the dehydrated reconstitutable polyacrylamide material.
  • the gel formation techniques of co-pending patent application U.S. Serial No. 08/209,632 which is invented by same inventor as in the present patent application, and which is incorporated herein by reference, are employed to initially prepare the polyacrylamide gel.
  • These include the use of a gel formation container which comprises a mixing bottle and a cap assembly including a compartment for storing pre-mixed ingredients for producing the polyacrylamide gel solution.
  • an acrylamide gel sample formation system comprising a leak-proof formation chamber including a pair of plates and a unitary U-shaped spacer member.
  • the chemical materials conventionally specified for producing an acrylamide gel solution are employed as the chemical formulation for producing the subject dehydrated reconstitutable polyacrylamide material.
  • the subject chemical formulation typically excludes a buffer salt but includes a stabilizer material.
  • the purpose of the stabilizer material is to maintain the crosslinking of the acrylamide and to retain the shape of the polyacrylamide gel during dehydration.
  • the stabilizer material typically comprises a material such as glycerol or sucrose.
  • the stabilizer material comprises from about 1% up to about 40% by weight, and more preferably from about 2% up to about 30% by weight, and most preferably about 5% up to about 20% by weight, of the total weight of the acrylamide gel solution.
  • the leak-proof formation chamber generally comprises a pair of plates each having a pair of major surfaces and a easily-alignable spacer member. The plates are assembled so that a major surface of each plate is adjacent to the other plate with the spacer member located between the plates thereby defining the formation chamber for casting polyacrylamide gel samples .
  • the acrylamide gel solution is introduced into the formation chamber.
  • the polyacrylamide gel sample is formed in the formation chamber in an intact condition.
  • the gel i ⁇ then either preserved in between cellophane membranes or laid on a screen and then dehydrated using air for 24 hours .
  • the reconstitutable polyacrylamide material in dehydrated form can be stored at ambient temperature for subsequent regeneration and use in, for example, electrophoretic analysis. More specifically, the dehydrated reconstitutable polyacrylamide material is typically storable at ambient temperature without loss of potency for at least about 120 days, preferably at least about 180 days, and more preferably at least about 300 days, and most preferably at least about 450 days .
  • the regeneration step can be preferably attained by providing a gel solution for regenerating the dehydrated reconstitutable polyacrylamide material. Then, this is accomplished by introducing the dehydrated reconstitutable polyacrylamide material into the gel solution and thereby regenerating the polyacrylamide gel sample for use in electrophoretic analysis.
  • the gel regenerating solution further preferably comprises a buffer salt, with or without a detergent.
  • a gel solution for regenerating the dehydrated reconstitutable polyacrylamide material is first formed.
  • Two pieces of fine mesh screen to be used as the gel formation system are employed for implementing gel regeneration.
  • the dehydrated material is placed in a shallow dish or the like which is filled with a gel regenerating solution.
  • the polyacrylamide will separate from the cellophane membrane, if present.
  • the first mesh screen is then placed on the bottom of the dish.
  • the polyacrylamide material is placed on top of the first mesh screen and is then covered with the second mesh screen.
  • the dish is shaken slowly on a rotary shaker. A 12% polyacrylamide gel sample is fully regenerated after 25 minutes.
  • the glass plate assembly is being prepared.
  • the first plate which typically is a large plate, is placed on the bench and a U-shaped spacer member is arranged on the top of the plate.
  • a PAGEMATETM U-shaped spacer manufactured by Essex Research of Portland, Oregon, is employed for this purpose.
  • the U-shaped spacer member comprises a horizontally-extending lower section and a pair of vertically-extending side sections of the U-shaped spacer member.
  • the fully regenerated gel is placed on the large plate, aligned with the inner dimension of the U-shaped spacer. Excess gel is then trimmed off the sides.
  • the ⁇ econd glass plate which is typically smaller in size, is placed on top of the U-shaped spacer member and the bottom of the glass plate assembly is clamped in position.
  • the gel-glass plate assembly is then clamped onto an electrophoresis apparatus.
  • the hold tab of the U-spacer is pulled to remove the bottom section and then the electrophoretic analysis is conducted in the usual manner .
  • the dehydrated reconstitutable material of the present invention can be formed from polyacrylamide gels produced as described in co-pending patent application U.S. Serial No. 08/209,632, as follows:
  • a polyacrylamide gel formulation typically comprises an acrylamide monomer solution (acrylamide and bis-acrylamide) of an appropriate strength, ammonium persulfate and TEMED.
  • the gel mixture additionally contains a stabilizer material, typically glycerol or sucrose, but no buffer salt .
  • B GEL CASTING.
  • a substantially leak-proof formation chamber system is typically employed for casting polyacrylamide gel samples.
  • the system includes a easily-alignable, unitary, substantially U- shaped spacer member and a pair of plates, which are generally made of glass.
  • the spacer member is formed from either silicon or plastic materials of different thickness.
  • the spacer member includes pull tabs for use in the subsequent removal of its bottom section after gel formation is completed.
  • the gel is casted according a predetermined size and thickness. After the polyacrylamide gel solidifies completely, the polyacrylamide gel formation as ⁇ embly i ⁇ taken apart and the gel is dehydrated in between ' cellophane membrane sheets so that it can be maintained in a pliable state. Alternatively, the gel is laid on a screen and is dehydrated in air.
  • ⁇ D) GEL REGENERATION To regenerate the dehydrated reconstitutable polyacrylamide material, the dehydrated reconstitutable polyacrylamide material together with the cellophane sheets is soaked in a gel regeneration solution.
  • the gel regeneration solution can comprise the user's choice of different buffer salts with or without a detergent such as SDS.
  • a preferred gel regeneration solution contains 0.375 M Tris.HCl (pH 8.8) and 0.1% SDS.
  • the cellophane sheets are separated from the polyacrylamide material and are removed from the buffer bath.
  • a gel holder Using a gel formation system ⁇ uch a gel holder will facilitate regeneration and handling of the gel in the gel regeneration solution. When the gel swells to size, it can then be taken out of the bath, re- assembled within the glass plates and then, onto electrophoresis apparatus. Employing a spacer member, preferably a U-shaped spacer member, facilitates the re-assembly process . The system is now ready for electrophoresis.
  • Example 1 This example shows a method of making a dehydrated reconstitutable polyacrylamide material sample, and a regenerated polyacrylamide gel sample produced therefrom.
  • Polyacrylamide gel samples were initially produced using a formation procedure, as follows: The gel were first prepared with the following stock solutions: A. Acrylamide/Bis 30% T, total monomers; 2.67
  • Cellophane sheets were cut to a size of 12 X 12 cm. Two sheets were needed for each polyacrylamide gel sample. The sheets were wetted briefly in a dish containing a solution of 12.5% glycerol. One sheet was laid over a glas ⁇ plate. The polyacrylamide gel was placed onto the cellophane sheet and it is then covered immediately with another sheet of cellophane. The excess cellophane is folded to the back around the glass plate and clamped in place. This assembly was dehydrated for 24 hours with air to form a dehydrated reconstitutable product. The dehydrated reconstitutable material together with the cellophane sheet was kept at room temperature for a period of at least about 450 days. Alternatively, the gel is laid on a screen to dehydrate same.
  • the gel shrinks to about 1/3 the original size and can be stored away in between protective plastic or paper sheets .
  • the dehydrated reconstitutable polyacrylamide material and the cellophane sheets or the shrunk dehydrated recon ⁇ tituted material were soaked in a gel regenerating solution.
  • the gel regenerating solution equilibrates and penetrates into the gel comprised buffer salts and SDS detergent.
  • a preferred gel regenerating solution contains 0.375 M Tris.HCl (pH 8.8) and 0.1% SDS.
  • the cellophane sheets, if present, were separated from the polyacrylamide material and were removed from the buffer bath.
  • a gel formation system including a gel holder was employed to facilitate regeneration and reswelling of the gel in the gel regenerating buffer.
  • the gel holder wa ⁇ porous. When the gel swelled to size, it was taken out of the bath, trimmed to size, re-as ⁇ embled within the glass plates and then introduced onto electro- phore ⁇ i ⁇ apparatus.
  • a U-shaped spacer member wa ⁇ employed to facilitate the re-a ⁇ embly proce ⁇ .

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Abstract

Matériau à base de polyacrylamide déshydraté, pouvant être reconstitué et destiné à être utilisé comme un échantillon de gel de polyacrylamide dans des analyses par électrophorèse. Ce matériau contient un matériau stabilisateur, mais ne contient pas de sel tampon. Il peut être stocké à température ambiante pendant au moins environ 120 jours sans subir de perte sensible d'activité.
PCT/US1995/015289 1995-11-27 1995-11-27 Materiaux a base de polyacrylamide pouvant etre reconstitues et procedes de production WO1997019989A1 (fr)

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PCT/US1995/015289 WO1997019989A1 (fr) 1995-11-27 1995-11-27 Materiaux a base de polyacrylamide pouvant etre reconstitues et procedes de production

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001050120A1 (fr) * 2000-01-03 2001-07-12 Gradipore Limited Procede de sechage de gels d'electrophorese

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4504641A (en) * 1982-02-19 1985-03-12 Fmc Corporation Polyacrylamide cross-linked with a polysaccharide resin as electrophoretic gel medium
US4746551A (en) * 1985-11-20 1988-05-24 Micro-Map, Inc. Rehydratable polyacrylamide gels
US5159049A (en) * 1991-04-22 1992-10-27 Allen Robert C Method for stabilizing polyacrylamide gels

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4504641A (en) * 1982-02-19 1985-03-12 Fmc Corporation Polyacrylamide cross-linked with a polysaccharide resin as electrophoretic gel medium
US4746551A (en) * 1985-11-20 1988-05-24 Micro-Map, Inc. Rehydratable polyacrylamide gels
US5159049A (en) * 1991-04-22 1992-10-27 Allen Robert C Method for stabilizing polyacrylamide gels

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001050120A1 (fr) * 2000-01-03 2001-07-12 Gradipore Limited Procede de sechage de gels d'electrophorese
US6472503B2 (en) 2000-01-03 2002-10-29 Gradipore Limited Method for drying electrophoresis gels

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