CA2243601A1 - Thermoreversible hydrogels comprising linear copolymers and their use in electrophoresis - Google Patents
Thermoreversible hydrogels comprising linear copolymers and their use in electrophoresis Download PDFInfo
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- CA2243601A1 CA2243601A1 CA002243601A CA2243601A CA2243601A1 CA 2243601 A1 CA2243601 A1 CA 2243601A1 CA 002243601 A CA002243601 A CA 002243601A CA 2243601 A CA2243601 A CA 2243601A CA 2243601 A1 CA2243601 A1 CA 2243601A1
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- copolymer
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- 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
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/52—Amides or imides
- C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
- C08F220/60—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing nitrogen in addition to the carbonamido nitrogen
- C08F220/606—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing nitrogen in addition to the carbonamido nitrogen and containing other heteroatoms
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F251/00—Macromolecular compounds obtained by polymerising monomers on to polysaccharides or derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F271/00—Macromolecular compounds obtained by polymerising monomers on to polymers of nitrogen-containing monomers as defined in group C08F26/00
- C08F271/02—Macromolecular compounds obtained by polymerising monomers on to polymers of nitrogen-containing monomers as defined in group C08F26/00 on to polymers of monomers containing heterocyclic nitrogen
Abstract
Thermoreversible hydrogels comprising non-ionic, linear copolymers, and methods of their use in electrophoresis, are provided. The subject copolymers comprise polyacrylamide backbones, where a portion of the acrylamide monomeric units comprise hydrogen bonding groups as N-substituents. Combination of the subject copolymers with an aqueous phase provides thermoreversible hydrogels which find use as separation media in electrophoretic applications.
Description
TEERMoREvl~R~ F. EYDROGELS COMPRISING L~AR
COPOLYMERS AND l ~;ll~ USE IN ELECTROPHORESIS
~TRODUCTION
5 Field of the ~-~vt;..Lion The ffeld of this invention is electrophoretic separation media.
.
R~rol m-l Electrophoresis has become an in~;leasi,lgly in~ pen~hle tool in 10 l~io~ nology and related fields. The ability to 5~s..~ molecules by means of size, shape and charge has added nuln~lvus opy~llulliLies to identify specific colllpuunds, de~l.uine purity, and allow for i~ol~tinn of a colllyound in a relatively pure form. A
variety of analytical techniques are preAic~t~d on the use of electrophoresis for the se.p~r~tiorl and analysis of the various components of interest that may be present in 15 a particular sample. For ~Y~m~le7 de~ ,yhoresis may be used to identify a colllyound~ where the coluponenls of a co..l?l~ ~ mixture are first s~a.d~d and then ~ubse~luelllly iflt~ntifi~d by using I~ Lt;.~ such as antibodies, DNA probes or the like. Ele.iLIoyllol~:,is may also be used in the d~ tic)n of the mo~ ul~r weights of colllyonenls in a sample.
Ele~LIoplloresis is usually pel~lllled in a sep~rAti-n media which provides for s~ u~ of the sample colnpollents as they migrate lhlough the gel under the infl~lence of an applied electric field. (~en~o~lly, sep~rAtion media which have found use in electrophoresis colnylise a network of either linear or cross-linked polymers.
~lth(.ugh a variety of different cross-linked and linear yolymers have been studied - for their suitability in cle~;L upl.~ ic appli~tion~, the most commonly employed ~lyll,e.~ are agarose and cross-linked po}yacrylamide.
Agarose gels, which comprise a linear ~ g co-polymer of ~
g71~rt~SP- and 3,6-anhydro-a-L-g~l~rt~se in an el~L,opl cs.~sis buffer, have many S advantages in el~L-~horesis. R~n~ they are thermoreversible, i.e. they undergoa t.rlh~;l;on from a first flowable state to second gel state in rP~ron~e to a change in te~ agarose gels are easy to prepare. Furthermore, agarose gels have high ",~ .,ir,~ LII~ providing for ease of manipulation. Another advantage of agarose gels is their ability to .s. ~., ~ large mol~ules, e.g. DNA from 200 bp to 10 about 50 kbp. Despite these ad~ ~es, there are disadvantages to the use of agarose gels as an cl~lluplloretic separation m~lium One disadvantage of agarosegels is their inability to provide for adequate r~solllfion of smaller sized colllpon~ ~It~. Other disad~ ~ges of agarose gels include the presence of gel isnpuriti~s that can result in sarnple co-~ ;or" distortions due to electroosmotic 1~ flow, and the like.
Cros~linkPd polyacrylamide gels, which are ~.~ through poly...~ ;on of acrylamide mcmnmPr with a cross-linker, provide ~lt~r~ ve separation media that overcome some of the problems ass~ ~t~i with agarose. Polyacrylamide gels provide for high resolution of small sized sample coll,pol,. r,~, e.g. they are capable 20 of providing high resolution of DNA ranging in size from 6 to L000 bp in length.
Other advantages of cross-linl~ed polyacrylamide gels are that: (1) they are optically ~ n~, providing for easy it1entifi~ ~tic~n of sep~L~d sample cc/~ ents, (2) they do not bind charged analytes and do not engenrl~r elc~;Lluo~.uotic flow, and (3) sample co.~.l~nent~ recovered from the gels are ~Ll~."ely pure, as the gels do not 2~ contain co~ as are found in agarose gels. Unfc,l L--"alely, since cross-linked poly~ la"lide gels must be l~l~cd in situ, their ~l~p~.At;on is comp~ tp~ and poses health risks, as the acrylamide mono,,,cl~ are toxic.
R~ll~ of the limit~tinn~ of the cUll~ nLly employed electrophoretic sep~r~tinn rnedia, for many cl~Ll~J~holc~ic applications it would be desirable to have 3û a gel which combined the high resolving power, as well as other adv~nt~ges, of cross-linked polyacrylamide with the thermoreversi~le nature of agarose.
WO 97/26288 PCT/US97/00'111 ~lPvs~nt T it~r5-tl-re Haas et al., J. Polym. Sci. B. (1964) 2: 1095, reports that poly(N-acrylyl~,lyc;~ les) form thermo~ ,il,le gels in which the transition telllp~alul~ of the gel rises with i~ t,al,ing col-~ e~ alion and molec~ r weight of the homopolymer.
S The copolylll~ aliol~ of N-acrylyl~ Iy~ le with acrylic acid, ~-aminoethyl vinyl ether, N-methacrylylvaline and iso~ opyla_.ylarnide was studied in Haas et aL, J.
Polym. Sci. A-2 (1967) 5:915 and Haas et al, J. Polym. Sci. A-l (1970) 8: 1131; 1213;
1725; and 3405. Haas et aL, J. Polym. Sci. A-1 (1971) 9:959 reported that solutions of poly(N m~,th~-;. ylylgly.~ es) gel upon cooling. Yoshioka et al., J. M.S. Pure Appl.
10 Chem. (1994) A31:113 repo~led the pr~ lion of aqueous sollltion~ of block copolymers of poly(N-isol,.u~ lacrylamide-co-n-butyl-mPth~rylate~ and poly(ethylene glycol), eYhi'r~ited reverse tr~nCiti~n l,~ ;1, ogel behavior in that solutions gelled upon heating.
Acrylylgly~ e homopolymers and their copolymers with acrolein or 15 methacrolein are reported in U.S. Pat. Nos. 3,452,182; 3,726,927 and 4,035,319.
ST T~ARY OF THF. I~VFI'ITION
Th~ ,o~ le hydrogels co",~ ,;.., linear copolymers are provided. The subject copolymers co-"~,i.,e pol~,~.c-yl~u-,-de bacl~ones in which a portion ofthe 20 acrylamide mol~n...~, jc units com~,ise l,y.l~ ,en bonding groups as N-s~.hstitl~Pntc By varying the nature of the copolymers, as well as the co~ ion of the copolyrners in the aqueous phase in which they are present, ll,G""o.~rersible hydrogels having a diverse range of physical characteristics are obtained. The subject the""o, e~fersible 1,yd-o~els find use as separation media in ele~,~,o~hol.:lic applications.
T~F!~CRrPTION OF TO TE~F. SPF.ClFIC Fl~oDrMFNTs Th~,.-"oit;~rersible hydrogels cc,.n~,i ,i"g copolymers are provided. The copolymers of the sub,ect hydrogels are nonionic and comprise a linear polyacrylamide backbone in which a po;tion ofthe acrylamide ...o--n...~-ic units comprise hydrogen 30 bondin 2, groups as N-substituent~ Upon con~hi~ n of the subject copolymers with an aqueous phase, a thermol.,~e.~,ible hydrogel is produced that ch~nge~ from a first, flowable state to a second, gel state over a narrow temperature range. The subject ,..ol~,.,rersible hydrogels find use as separaeion media in ele~ ophon,l.c appi~ ;onc In further des~i,ibih~g the sub}ect invention, the copoiymers w-vill be de3_.il,ed first in greater detail followed by a description of Lhe.l~ol~ve.s;blc gels Co~ JI;..;llg the subject copolymers, as well as their use in elccllopho~ ic ~l~pli"~,l;on~
The copolymers of the subject i,l~.. ,Lion are linear, non-ionic copolymers cc...~ g a poly~l;,yl~nide backbone, where a portion of the acrylamide ...OI-- ,n. . ic units CO~ ,l i ,c N-substih~Pnt groups capable of hydrogen bol~Jill~;. The mohc~ r weight ofthe subject polymers will be at least about 10 kD, more usually at least about 50 kD, and may be as high as 1000 Id~ or higher. The term acrylamide as used herein 10 in~ ec ....~l.s~ acrylam-de and deliva~ s thereof, such as rn~th~..lylâ~llide, and the like, as well as N-substituted derivatives thereof. The weight percent ratio of acryiamide monomeric units of the copolymer colllp.i~ g N-s-lbstituent groups capable of hydrogen bonding will range from about 55:45 to 95 :5, and will usually range from about 65:35 to 90:10.
The hydrogc.~ bonding N-s~1bstitl1ent groups ofthe copolymers will COm~ e a hrd~u~,cn bonding moiety bonded through a bond or linking group to the N atom of the a~,,ylalnide ....~--o...- . ic unit. The copolymers of the subject invention may be homogeneous as to the nature of the hydrogen bonding N-sllhstit~1ent group, or heterogeneous, colll~,li:,illg up to 6 di~ere..~ hydrogen bondillg N-substituent groups, 20 but will usually comprise no more than 4 hydrogen bon.lh~g N-s~lbstituent groups, more usually no more 2 hydrogen bondill~ N-s-~hstitnent groups. The hydrogen bonding N-substit~ent group will be capable of ~Ill~ing inter- and intramolecular hydrogen bonds in an aqueous ~eJ;~ and will colll~JIise from 2 to 30 carbon atoms, usually from 2 to 20 carbon atoms, more usually from 2 to 10 carbon atoms, and may be aliphatic, 25 ali~;y-,lic, an,..,alic or heterocyclic, particularly ~lirl~tic or heterocyclic. The hydrogen bondll~g moiety of the group will generally be a carbamyl moiety. Particular s~lhstitt~ent groups of interest include heterocyclic ,li~,ogell bases, where the nitrogen is ~u13~ ly neutral at neutral pH, arnides, particularly aliphatic amides, and the like.
Hct~,ro~;yclic ,li~.ogc.l bases of interest include: purines, such as ~-~nin~ nin~
30 I~y~ Y~ ; py~ ei~ such as thymine, cytosine, inosine, uracil; as well as natural and synthetic mitn~tics thereof. Arnides of interest will be a to the N of the acrylamide ic unit, and will include ~lirh~tis amides, where the aliphatic portion of the - ~liph~ti(~ amide will range from 1 to 4 carbon atoms, usually -1 to 3 carbon atoms, more usually 1 to 2 carbon atoms.
The copolymers of the sub~ect invention will be conveniently pl e~,&. èd from first and second monomP~rs~ ~irst ,-,ono,l,cl~ that find use in the subject invention may be cle,~ ~1 by the formula:
X O
l 11 wl,~,rei-l:
X is H or CH3, Y is a bond or a linking group, where the linking group may be an ~liphntic chain of from 1 to 6 carbon atoms, usually lto 4 carbon atoms, more usually 1 to 2 carbonatoms, where the ~liph~tic chain may be a straight or branched chain, comprising from 0 to 2 sites of unsa~ulalion; and Z is a youp CCI--p.i:,l"g a hydrogen bonding moiety, where Z may be: from 2 to 30 carbon atoms, usually from 2 to 20 carbon atoms, more usually from 2 to 10 carbon atoms; will cG",plise from 2 to 10 hcleroàlollls~ usually 2 to 8 heteroatoms, where at least one of the helelualoms wi11 be an N bonded to an H; and may be aliphatic, alicyclic, aromatic or heterocyclic, particularly aliphatic or heterocyclic, comprising 0 to 3 ring structures, usuaily 0 to 2 ring structures, where the ring structures may be fused and will generally be 5 to 6 atom rings.
The hydrogen bonding moiety present in the Z group will generally be a carbamyl group, where calbà~ l group as used herein is described by the formula: . D
A--c--NH
A is C or a hel~,ro~Lulll;
30 D is O or S, usually O; and Rl is H or an ~liph~tic s~sliluclll of up to 10 carbon atoms, usually up to 6 carbon atoms, more usually up to 4 carbon atoms, where the alkyl substituent may be straight or bl~-cl-ed chain.
W}iere Z is an ~lirh~tic. amide, the first Illonolllc. will have the formula:
X O O
H2C=C--C--NH--Y--C--NRl}I
W~
5 X and Y and Rl are the same as defined above;
Specific l--OI-O~ , that find use as first mc notners in the subject invention include N-s~.l"~ d acrylamide and the like. P.~ bly, the first ....~ will be acrylylglyc;.~-... le The subject ,..~nn~ " ~ may be pr~l.~ed accol di.~g to known metho~1c~ such as those de.,_-il,ed in U.S. Pat. No. 3,452,182 for the p~pal~Lion of 10 amino s.lb~ -led ~liph~ti~ amides, the ~liC.,lo~ ..c; of which is herein i~colyo~aled by nce.
The second monomer, which is copolyl,.~ .,d with the first monomer to produce the subject copolymers, will be ac,.yl~. idc. Any N-substituf. ntc of the second l,.ono...cl will not comprise a hydrogen bonding moiety. Preferably, the second 15 Illol~o~lf ~ will be llnc~bstitlltec~ acrylamide.
The subject copolymers may be prepdl~d acco,~lil~ to known methods by co., .l~ g the proper ratio of first and second ~..ono.-.~ in a fluid phase and i~ g poly...~, iLalion. The ratio of first to second monom~r which is co.--bined in the aqueous phase will depend, in part, on the desired p, upc. lies of the thermoreversible gel which 20 is p~ d from the copolymer, e.g the desired melting tt;...p~ L~- e range at which a hydrogel CGInpl i.,;ilg the copolymer will change from a gel to a flowable solution. Thus, if a l.y.l. ogel with a high melting t~,.ll?.,. aLul c range is desired, the ratio of first to second monomers which are combined and co-polyl.lt;li,ed will be high. Alternatively, the ratio of first to second monomers will be low if thermoreversible gels having a 25 lower melting tell~ aL~re range are desired. Generally, the mole ratio of first to second monol..~ will range from about 45:5~ to 95:~, more usually from about 50:50 to 90:10.
The fluid phase employed for poly...~. i~Lion may be an aqueous or non-aqueous phase. A variety of aqueous phases may be employed, inch!-1ing pure water 30 and water/}ower alkanol mixtures, where the lower aLkanol will typically be a C4 or smaller alkanol, such as ethanol, propanol, iso~.ul,yl alcohol and the like. Instead of, or in addition to, a lower alkanol, other polar organic solvents may be employed as co-solvents, such as dil-~Ll~yl~Ol.~ iç~ dimethylsulfoxide and the like. The volumepercent of the water in the aqueous phase will range from 10 to 100 %. The volume - percent of the co-solvent, when present, in the aqueous phase will not exceed 90%, and will usually not exceed 50 %. A non-aqlleol-s phase may also be employed, where the non-aqueous phase may be any convenient organic solvent, such as those listed above.
In some incl ~nCr C where the resultant copolymer and pol-y-~~ç~ n fluid phase S are to be used directly as a separation media for ele~,lfoph/sres;s, it may be COIl~/tl~_ to include ~cln'ition~l agents in the flluid phase which find use in elecL.upholcs;s.
Additional agents of interest include various salts, particularly buffering salts, where the col~r~ tion ofthe buffering salts will vary from 0.01 to 0.5, more usually firom 0.01 to 0. l M. The salts may include Tris, phosrh~te~ EDTA, MOPS, and the like.
10 Dellalulill~3, agents may also be present in the aqueous phase, patticularly where the aqueous phase present during copoly---e-i~Lion will also serve as the contim~ous fluid phase in the hydrogel during elc~,l-o~ho-~.is. De~laLulillg agents that may be present in the aqueous phase include urea, SDS, r... ~ le, methylmercuric hydroxide, alkali, and the like, where the concel~ lion will vary depc..~i.Zg on the particular denaturing 15 agent, e.g. for urea, the concc..ll~Lion will range from about 0.1 to 9.0 M.
Poly-..~.i~Lion may be in,~ ed using any convenient means, in~ rling both physical and ~.h,.~-ral means. Physical means that may be employed include exposure to ultrasound, ultraviolet light and y-ray irradiation. Chemical initiators that may be empioyed include: persl-lr.h~te + 3-din.~,lhylaminopropioniLIile (DMPAN), persulphate 20 + tct-a-~-~,Ll-ylethyl- -~PAi~n~ r (TEMED), ~ haLe + heat, persulphate ~ thioslllf~te, persulphate + bisulfiite, pc.~ lph~(e + .I;.,~ I--Iethyi~n.;"çA;,~ ç ~DEMED), H2O2 +
Fe2~, benzoyl peroxide, lauroyl peroxide, tetralin peroxide, actyl peroxide, caproyl peroxide, t-butyl hy~Lu~Je~ ide, t-butyl p~,.l,Fn~v~e t-butyl diperph~h~l~tç cumene l~ydlu~ero~ide, 2-bu~dllone peroxide, ~o;~ tor.., e.g. azodiisobutylnitrile and 25 ~7,n~;r~-1,on~,--de, riboflavin + visible light, methylene blue + a redox couple, and the 1ike. Pr~,f~.~ly a ~h~m;c~l polymerization initiator such as pe. tul~ha~e will be employed. When necç ,,j~ . y to limit exposure of the monomers to oxygen during poly..,e,i~aLion, pol~",~ ion may be carried out in an oxygen free atmosphere, such a n i~lc.~ l al",o;",he.e.
Following poly"l~;-i~ion, the resultant copolymers in co-"bi.. dLion with a fluid phase can be used directly as a separation n.edi~ l for dcoL.o~uhoresis, where the concf~ Lion of the copolymers in the fluid phase provides for a hydrogel having WO 97/26288 PCTtUS97/00411 - 1 - u~ t~es suitable for use in eleu~ opholeD;s, or the copolymers can be sel,a- ~L~,d from the fluid phase and stored until later use, as appropliale. The copolymers can be recovered from the fluid phase using any convenient means, such as freeze drying or To prepare thermoreversible hydrogels from the subject copolymers, a sufficient amount of copolymer will be co~ ;..ed with an aqueous me~i-lm, where the aqueousprovides the continllQus fluid phase ofthe hydrogel. Generally, the amount of copol~ f that is co...l.i~fd with the a~ eouC ."~1;..,., will range from about 1 to 30 %T, and will usually range from about 2 to 20 %T, more usually from about 3 to 15 lû %T, where %T refers to the total weight of copolymer in grams per 100 ml of aqueous meriillm As described above, the aqueous mP~ m may comprise various agents that find use in elc~,~, uphoi e.,is, such as l>u~. ing salts, denaturing agents, and the like.
The subject th~ ,ol~,.tcrsible hydrogels are cl-~cle-~ed by undergoing a D~bD~ physical change over a narrow melting le...~ u-e range (Tm)~ where below 15 the Tm the hydrogel is present as a gel like composition having a high viscosity and capable of ele~,~-ophoretic sieving. Above the Tm of the hydrogel, the hydrogel is present as a flowable, pourable cu...poD;lion having a low viscosity and being h~capablc of ele~,L~upho.e~ic sieving. The Tm of a particular hydrogel accc.dil-g to the subject invention, as well as the physical p. upe~ Lies of the hydrogel above and below the Tm~
will depend on the both the nature of the particular copolyrner from which the gel is p-~ d, as well as the concel.L.~lion of the copolymer in the aqueous phase.
Generally, the subject hydrogels will have a Tm between about 5 and 80~C, usually bt:t~.ecn about 10 and 70 ~C, and more usually between about 15 and 65 ~C. The m~gnit~lde ofthe narrow range ofthe Tm will range from about 0.1 to 10 ~C, and will usually range from a-hout 0.1 to 7.5 ~C, more usually from ahout 0.1 to 5.0~C. Above the Tm~ the subject hydrogels will have a ~risco:,;ly ranging from about 5 to 30,000 cps, more usually from about 20 to 10,000 cps, while below the Tm the subject l"rdlog~,ls will have a sllhst~nti~lly infinite viscosity.
dition to the subiect copolymers described above, the the, ~l~u~ ersible hydrogels may further comprise one or more additional, non-p.ot~ ceQ~Is~ polymers that serve to mo(~ te the physical and/or sieving characteristics of the hydrogel. These ~clrlitic~nS~I polyrners may be linear or b-anclled, and include hydroxyethylc~ llos~
- ily~lluAy~ yyl cpllnlos~ methyl cf llulosç, llydlu~y~JIo~Jyl methyl cfAIIt~lose, polyethylene glycol, polyethylene oxide, galacto ..~I~nAll pullulan, dextran, polyvinyl alcohol, agarose, polyacryloylamino-etho~y~;lhanol and the like The weight pw~,e,-lage of these arl~tition~l polymers present in the hydrogel per milltlifPr of a~ueous phase will 5 depend on the particular ~ ition~l poly",er, the copolymer and the desired chara~ lics of the hydrogel. Generally, the T of the additional polymer or polymers in the hydrogel will range from 0 1 to 5.0 %, more usually from 0 1 to 2.0 %
The subject ll~.",u~ rsi~le Lydlogf ls find use as separation media for ele~l-ùphoretic appli~ ~;on~ Elecllophol~t;c a~plic~l;Qn~ in which the subject 10 hydrogels find use as separation media are well known, being des~i,il,ed in Andrews, Ele~ ophu~ s (1986) and Barron & Blanch, Separation and Purification Methods (1995) 24:1-118, and need not be reviewed in great detail here Briefly, in e le~,l.ophu-f .ic appli--,.t;onc, the subject media will be placed in an electrophoretic separation chal.~ , e.g a slab gel CO~ f l, colurnn, t h~nnçl, capillary and the like, of 15 an ele~l,ùpho~Lic device Although the cleclrophoresis device can be pl~,tJâl~,;l by producing the the"nol~ ersible hydrogel in situ, typically a pre-p,e,oa,Gd hydrogel will be introduced into the ele~ ùphGl ~lic ~hallll)el ~ where the hydrogel is in the first, flowable state Thus, the hydrogel may be pr-_p&r~d as described above and then introduced into the separation ~h~mbPr of the electrophoretic device when the 20 te"~p."al.lre of the hydrogel is above the Tm~ i.e. as a pregel solution. After being introduced into the ;,cpa, ~lion .,h~,.l.er, the te...~ re of the hydrogel may then be lowered so that the hydrogel ~nm~os a gel state, capab}e of elccl,ophoretic sieving The hydrogel may be introduced into the separation chan,be, using any convenientmeans Thus, for slab gel holders, it may be s~;cient to simply pour the pregel solution 25 into the s1ab gel holder while the hydrogel is above the Tm. Alternatively, for capillary holders, it may be more convenient to introduce the gel, while in a fluid state, into the interior volume of the capillary through inje~,lion or suction.
Once the hydrogel has been introduced into the separation çh~mh~r of the elecll ~,phur~,lic device and the lt~ pe~lufe of the hydrogel reduced to below the Tm~ a 3Q sample may be introduced into the hydrogel for ele-,l,opho,~,i,;s Where convenient, the l,~d,ogel may be prc-ele~,Ln)phoresed, where pre-electrophoresis can serve a variet,v of ~U11303eS, such as for introduction of se~JalaLion buffer, and the like Sample -- components which may be separated in the subject hydrogels include nucleic acids, IJlOt,~,;lls, carbohydrates and the like. The sample may be introduced into the gel using a variety of methods, with the particular method sPlected dependent on the type of device being e.l~loyed. Electrophoresis of the sample in the hydrogel may then be carried out S in accol~ ce with known procedures.
Following ~lecl.ophGlc,~,;s, the separated sample components may be analyzed in the gel, e.g by staining and the like. The ele.,~ horetically se~ala~ed sample cc,.llpon~;llL~. may be ~-,.lloved from the gel for further analysis. Depen1i.lg on the part;cular ele~l-opholc~lic device being e.~loyed, separation may be accGlll~lished by 10 blotting, or by raising the tellli)e.aLu.~ ofthe hydrogel above the Tm and then extracting the sample co...pol1~,..L of the interest from the resultant fluid me~ m In addition to their use in cle.,~lupholes.s, the subject copolymers find use inother separation applications such as ~ ollla~ography, as well as in lllelllbl ~nes, controlled release compocitiorlc~ contact lenses, and the like.
The following PY~mples are offered by way of illustration and not by way of limit~fi- n.
~;.X~ K~ AT.
A number of dirrel ~ copolymers of acrylylglycin~m;~e and acrylamide were p. ~ ,d and their characteristics were colll~ared to homopolymers of polyacrylylgly~ le 25 A. Gels of Acrylylglycin~ e and Acrylamide Copolymers ~AGA) 1. T8% Copolymer AGA50 A copolymer of acrylyl~,ly~ ,.... de (AG) and acrylamide (AA) was plepaled by collllfilllng AG and AA mono.nGl~ in pure water at room tel.lp~,.al-lre with ~lln OlU~
pers~llph~te (APS) and N,N,N'N'-te~.a..lcLl.ylethylçneAi~mine (TEMED) as 30 polylll.,.i~aliOn illiLiaLOl :,. The weight percent ratio of AG to AA monomers was 50:~0.
The conf~ .aLion oftotal monomer prior to pol~llle~i~aLion was 8 %. Upon - polylllc. i~,aliOl~ at room ten~pGl ~ re, a solution of a linear viscous polymer was ol~lailled.
2 T7.3% Copolymer AGA 73 The procedure used to produce the T8 AGA50 was employed, except that the ratio weight percent of AG to AA was Ghànged to 73 :27, and the conce.,ll i.lion of total nolne. prior to poly.,~ ;alion was 7.3 %. Following poly...~ ion at room tc.ll~e.~L~ t a Il~G~ or~rcrsible clear gel was obtained. The resultant hydrogel had a T~"
of 36.5 ~C.
3. T6.4% Copolymer AGA90 The same procedure as used to prepare the T7.3%AGA73 gel was employed, except that the weight % ratio of AG to AA was rh~nged to 90:10 and the COllC~ lLlalion of total ",onol~er prior to polymerization was 6.4%. Poly",~ Lion yielded a l~ pu~,-,L gel at room te.ll~ lulG which had a Tm of 63.9 ~C. The resultant T6.4% AGA90 gel was observed to be m~ch~r~je~lly stronger and more stable than the T7.3% AGA73 gel.
COPOLYMERS AND l ~;ll~ USE IN ELECTROPHORESIS
~TRODUCTION
5 Field of the ~-~vt;..Lion The ffeld of this invention is electrophoretic separation media.
.
R~rol m-l Electrophoresis has become an in~;leasi,lgly in~ pen~hle tool in 10 l~io~ nology and related fields. The ability to 5~s..~ molecules by means of size, shape and charge has added nuln~lvus opy~llulliLies to identify specific colllpuunds, de~l.uine purity, and allow for i~ol~tinn of a colllyound in a relatively pure form. A
variety of analytical techniques are preAic~t~d on the use of electrophoresis for the se.p~r~tiorl and analysis of the various components of interest that may be present in 15 a particular sample. For ~Y~m~le7 de~ ,yhoresis may be used to identify a colllyound~ where the coluponenls of a co..l?l~ ~ mixture are first s~a.d~d and then ~ubse~luelllly iflt~ntifi~d by using I~ Lt;.~ such as antibodies, DNA probes or the like. Ele.iLIoyllol~:,is may also be used in the d~ tic)n of the mo~ ul~r weights of colllyonenls in a sample.
Ele~LIoplloresis is usually pel~lllled in a sep~rAti-n media which provides for s~ u~ of the sample colnpollents as they migrate lhlough the gel under the infl~lence of an applied electric field. (~en~o~lly, sep~rAtion media which have found use in electrophoresis colnylise a network of either linear or cross-linked polymers.
~lth(.ugh a variety of different cross-linked and linear yolymers have been studied - for their suitability in cle~;L upl.~ ic appli~tion~, the most commonly employed ~lyll,e.~ are agarose and cross-linked po}yacrylamide.
Agarose gels, which comprise a linear ~ g co-polymer of ~
g71~rt~SP- and 3,6-anhydro-a-L-g~l~rt~se in an el~L,opl cs.~sis buffer, have many S advantages in el~L-~horesis. R~n~ they are thermoreversible, i.e. they undergoa t.rlh~;l;on from a first flowable state to second gel state in rP~ron~e to a change in te~ agarose gels are easy to prepare. Furthermore, agarose gels have high ",~ .,ir,~ LII~ providing for ease of manipulation. Another advantage of agarose gels is their ability to .s. ~., ~ large mol~ules, e.g. DNA from 200 bp to 10 about 50 kbp. Despite these ad~ ~es, there are disadvantages to the use of agarose gels as an cl~lluplloretic separation m~lium One disadvantage of agarosegels is their inability to provide for adequate r~solllfion of smaller sized colllpon~ ~It~. Other disad~ ~ges of agarose gels include the presence of gel isnpuriti~s that can result in sarnple co-~ ;or" distortions due to electroosmotic 1~ flow, and the like.
Cros~linkPd polyacrylamide gels, which are ~.~ through poly...~ ;on of acrylamide mcmnmPr with a cross-linker, provide ~lt~r~ ve separation media that overcome some of the problems ass~ ~t~i with agarose. Polyacrylamide gels provide for high resolution of small sized sample coll,pol,. r,~, e.g. they are capable 20 of providing high resolution of DNA ranging in size from 6 to L000 bp in length.
Other advantages of cross-linl~ed polyacrylamide gels are that: (1) they are optically ~ n~, providing for easy it1entifi~ ~tic~n of sep~L~d sample cc/~ ents, (2) they do not bind charged analytes and do not engenrl~r elc~;Lluo~.uotic flow, and (3) sample co.~.l~nent~ recovered from the gels are ~Ll~."ely pure, as the gels do not 2~ contain co~ as are found in agarose gels. Unfc,l L--"alely, since cross-linked poly~ la"lide gels must be l~l~cd in situ, their ~l~p~.At;on is comp~ tp~ and poses health risks, as the acrylamide mono,,,cl~ are toxic.
R~ll~ of the limit~tinn~ of the cUll~ nLly employed electrophoretic sep~r~tinn rnedia, for many cl~Ll~J~holc~ic applications it would be desirable to have 3û a gel which combined the high resolving power, as well as other adv~nt~ges, of cross-linked polyacrylamide with the thermoreversi~le nature of agarose.
WO 97/26288 PCT/US97/00'111 ~lPvs~nt T it~r5-tl-re Haas et al., J. Polym. Sci. B. (1964) 2: 1095, reports that poly(N-acrylyl~,lyc;~ les) form thermo~ ,il,le gels in which the transition telllp~alul~ of the gel rises with i~ t,al,ing col-~ e~ alion and molec~ r weight of the homopolymer.
S The copolylll~ aliol~ of N-acrylyl~ Iy~ le with acrylic acid, ~-aminoethyl vinyl ether, N-methacrylylvaline and iso~ opyla_.ylarnide was studied in Haas et aL, J.
Polym. Sci. A-2 (1967) 5:915 and Haas et al, J. Polym. Sci. A-l (1970) 8: 1131; 1213;
1725; and 3405. Haas et aL, J. Polym. Sci. A-1 (1971) 9:959 reported that solutions of poly(N m~,th~-;. ylylgly.~ es) gel upon cooling. Yoshioka et al., J. M.S. Pure Appl.
10 Chem. (1994) A31:113 repo~led the pr~ lion of aqueous sollltion~ of block copolymers of poly(N-isol,.u~ lacrylamide-co-n-butyl-mPth~rylate~ and poly(ethylene glycol), eYhi'r~ited reverse tr~nCiti~n l,~ ;1, ogel behavior in that solutions gelled upon heating.
Acrylylgly~ e homopolymers and their copolymers with acrolein or 15 methacrolein are reported in U.S. Pat. Nos. 3,452,182; 3,726,927 and 4,035,319.
ST T~ARY OF THF. I~VFI'ITION
Th~ ,o~ le hydrogels co",~ ,;.., linear copolymers are provided. The subject copolymers co-"~,i.,e pol~,~.c-yl~u-,-de bacl~ones in which a portion ofthe 20 acrylamide mol~n...~, jc units com~,ise l,y.l~ ,en bonding groups as N-s~.hstitl~Pntc By varying the nature of the copolymers, as well as the co~ ion of the copolyrners in the aqueous phase in which they are present, ll,G""o.~rersible hydrogels having a diverse range of physical characteristics are obtained. The subject the""o, e~fersible 1,yd-o~els find use as separation media in ele~,~,o~hol.:lic applications.
T~F!~CRrPTION OF TO TE~F. SPF.ClFIC Fl~oDrMFNTs Th~,.-"oit;~rersible hydrogels cc,.n~,i ,i"g copolymers are provided. The copolymers of the sub,ect hydrogels are nonionic and comprise a linear polyacrylamide backbone in which a po;tion ofthe acrylamide ...o--n...~-ic units comprise hydrogen 30 bondin 2, groups as N-substituent~ Upon con~hi~ n of the subject copolymers with an aqueous phase, a thermol.,~e.~,ible hydrogel is produced that ch~nge~ from a first, flowable state to a second, gel state over a narrow temperature range. The subject ,..ol~,.,rersible hydrogels find use as separaeion media in ele~ ophon,l.c appi~ ;onc In further des~i,ibih~g the sub}ect invention, the copoiymers w-vill be de3_.il,ed first in greater detail followed by a description of Lhe.l~ol~ve.s;blc gels Co~ JI;..;llg the subject copolymers, as well as their use in elccllopho~ ic ~l~pli"~,l;on~
The copolymers of the subject i,l~.. ,Lion are linear, non-ionic copolymers cc...~ g a poly~l;,yl~nide backbone, where a portion of the acrylamide ...OI-- ,n. . ic units CO~ ,l i ,c N-substih~Pnt groups capable of hydrogen bol~Jill~;. The mohc~ r weight ofthe subject polymers will be at least about 10 kD, more usually at least about 50 kD, and may be as high as 1000 Id~ or higher. The term acrylamide as used herein 10 in~ ec ....~l.s~ acrylam-de and deliva~ s thereof, such as rn~th~..lylâ~llide, and the like, as well as N-substituted derivatives thereof. The weight percent ratio of acryiamide monomeric units of the copolymer colllp.i~ g N-s-lbstituent groups capable of hydrogen bonding will range from about 55:45 to 95 :5, and will usually range from about 65:35 to 90:10.
The hydrogc.~ bonding N-s~1bstitl1ent groups ofthe copolymers will COm~ e a hrd~u~,cn bonding moiety bonded through a bond or linking group to the N atom of the a~,,ylalnide ....~--o...- . ic unit. The copolymers of the subject invention may be homogeneous as to the nature of the hydrogen bonding N-sllhstit~1ent group, or heterogeneous, colll~,li:,illg up to 6 di~ere..~ hydrogen bondillg N-substituent groups, 20 but will usually comprise no more than 4 hydrogen bon.lh~g N-s~lbstituent groups, more usually no more 2 hydrogen bondill~ N-s-~hstitnent groups. The hydrogen bonding N-substit~ent group will be capable of ~Ill~ing inter- and intramolecular hydrogen bonds in an aqueous ~eJ;~ and will colll~JIise from 2 to 30 carbon atoms, usually from 2 to 20 carbon atoms, more usually from 2 to 10 carbon atoms, and may be aliphatic, 25 ali~;y-,lic, an,..,alic or heterocyclic, particularly ~lirl~tic or heterocyclic. The hydrogen bondll~g moiety of the group will generally be a carbamyl moiety. Particular s~lhstitt~ent groups of interest include heterocyclic ,li~,ogell bases, where the nitrogen is ~u13~ ly neutral at neutral pH, arnides, particularly aliphatic amides, and the like.
Hct~,ro~;yclic ,li~.ogc.l bases of interest include: purines, such as ~-~nin~ nin~
30 I~y~ Y~ ; py~ ei~ such as thymine, cytosine, inosine, uracil; as well as natural and synthetic mitn~tics thereof. Arnides of interest will be a to the N of the acrylamide ic unit, and will include ~lirh~tis amides, where the aliphatic portion of the - ~liph~ti(~ amide will range from 1 to 4 carbon atoms, usually -1 to 3 carbon atoms, more usually 1 to 2 carbon atoms.
The copolymers of the sub~ect invention will be conveniently pl e~,&. èd from first and second monomP~rs~ ~irst ,-,ono,l,cl~ that find use in the subject invention may be cle,~ ~1 by the formula:
X O
l 11 wl,~,rei-l:
X is H or CH3, Y is a bond or a linking group, where the linking group may be an ~liphntic chain of from 1 to 6 carbon atoms, usually lto 4 carbon atoms, more usually 1 to 2 carbonatoms, where the ~liph~tic chain may be a straight or branched chain, comprising from 0 to 2 sites of unsa~ulalion; and Z is a youp CCI--p.i:,l"g a hydrogen bonding moiety, where Z may be: from 2 to 30 carbon atoms, usually from 2 to 20 carbon atoms, more usually from 2 to 10 carbon atoms; will cG",plise from 2 to 10 hcleroàlollls~ usually 2 to 8 heteroatoms, where at least one of the helelualoms wi11 be an N bonded to an H; and may be aliphatic, alicyclic, aromatic or heterocyclic, particularly aliphatic or heterocyclic, comprising 0 to 3 ring structures, usuaily 0 to 2 ring structures, where the ring structures may be fused and will generally be 5 to 6 atom rings.
The hydrogen bonding moiety present in the Z group will generally be a carbamyl group, where calbà~ l group as used herein is described by the formula: . D
A--c--NH
A is C or a hel~,ro~Lulll;
30 D is O or S, usually O; and Rl is H or an ~liph~tic s~sliluclll of up to 10 carbon atoms, usually up to 6 carbon atoms, more usually up to 4 carbon atoms, where the alkyl substituent may be straight or bl~-cl-ed chain.
W}iere Z is an ~lirh~tic. amide, the first Illonolllc. will have the formula:
X O O
H2C=C--C--NH--Y--C--NRl}I
W~
5 X and Y and Rl are the same as defined above;
Specific l--OI-O~ , that find use as first mc notners in the subject invention include N-s~.l"~ d acrylamide and the like. P.~ bly, the first ....~ will be acrylylglyc;.~-... le The subject ,..~nn~ " ~ may be pr~l.~ed accol di.~g to known metho~1c~ such as those de.,_-il,ed in U.S. Pat. No. 3,452,182 for the p~pal~Lion of 10 amino s.lb~ -led ~liph~ti~ amides, the ~liC.,lo~ ..c; of which is herein i~colyo~aled by nce.
The second monomer, which is copolyl,.~ .,d with the first monomer to produce the subject copolymers, will be ac,.yl~. idc. Any N-substituf. ntc of the second l,.ono...cl will not comprise a hydrogen bonding moiety. Preferably, the second 15 Illol~o~lf ~ will be llnc~bstitlltec~ acrylamide.
The subject copolymers may be prepdl~d acco,~lil~ to known methods by co., .l~ g the proper ratio of first and second ~..ono.-.~ in a fluid phase and i~ g poly...~, iLalion. The ratio of first to second monom~r which is co.--bined in the aqueous phase will depend, in part, on the desired p, upc. lies of the thermoreversible gel which 20 is p~ d from the copolymer, e.g the desired melting tt;...p~ L~- e range at which a hydrogel CGInpl i.,;ilg the copolymer will change from a gel to a flowable solution. Thus, if a l.y.l. ogel with a high melting t~,.ll?.,. aLul c range is desired, the ratio of first to second monomers which are combined and co-polyl.lt;li,ed will be high. Alternatively, the ratio of first to second monomers will be low if thermoreversible gels having a 25 lower melting tell~ aL~re range are desired. Generally, the mole ratio of first to second monol..~ will range from about 45:5~ to 95:~, more usually from about 50:50 to 90:10.
The fluid phase employed for poly...~. i~Lion may be an aqueous or non-aqueous phase. A variety of aqueous phases may be employed, inch!-1ing pure water 30 and water/}ower alkanol mixtures, where the lower aLkanol will typically be a C4 or smaller alkanol, such as ethanol, propanol, iso~.ul,yl alcohol and the like. Instead of, or in addition to, a lower alkanol, other polar organic solvents may be employed as co-solvents, such as dil-~Ll~yl~Ol.~ iç~ dimethylsulfoxide and the like. The volumepercent of the water in the aqueous phase will range from 10 to 100 %. The volume - percent of the co-solvent, when present, in the aqueous phase will not exceed 90%, and will usually not exceed 50 %. A non-aqlleol-s phase may also be employed, where the non-aqueous phase may be any convenient organic solvent, such as those listed above.
In some incl ~nCr C where the resultant copolymer and pol-y-~~ç~ n fluid phase S are to be used directly as a separation media for ele~,lfoph/sres;s, it may be COIl~/tl~_ to include ~cln'ition~l agents in the flluid phase which find use in elecL.upholcs;s.
Additional agents of interest include various salts, particularly buffering salts, where the col~r~ tion ofthe buffering salts will vary from 0.01 to 0.5, more usually firom 0.01 to 0. l M. The salts may include Tris, phosrh~te~ EDTA, MOPS, and the like.
10 Dellalulill~3, agents may also be present in the aqueous phase, patticularly where the aqueous phase present during copoly---e-i~Lion will also serve as the contim~ous fluid phase in the hydrogel during elc~,l-o~ho-~.is. De~laLulillg agents that may be present in the aqueous phase include urea, SDS, r... ~ le, methylmercuric hydroxide, alkali, and the like, where the concel~ lion will vary depc..~i.Zg on the particular denaturing 15 agent, e.g. for urea, the concc..ll~Lion will range from about 0.1 to 9.0 M.
Poly-..~.i~Lion may be in,~ ed using any convenient means, in~ rling both physical and ~.h,.~-ral means. Physical means that may be employed include exposure to ultrasound, ultraviolet light and y-ray irradiation. Chemical initiators that may be empioyed include: persl-lr.h~te + 3-din.~,lhylaminopropioniLIile (DMPAN), persulphate 20 + tct-a-~-~,Ll-ylethyl- -~PAi~n~ r (TEMED), ~ haLe + heat, persulphate ~ thioslllf~te, persulphate + bisulfiite, pc.~ lph~(e + .I;.,~ I--Iethyi~n.;"çA;,~ ç ~DEMED), H2O2 +
Fe2~, benzoyl peroxide, lauroyl peroxide, tetralin peroxide, actyl peroxide, caproyl peroxide, t-butyl hy~Lu~Je~ ide, t-butyl p~,.l,Fn~v~e t-butyl diperph~h~l~tç cumene l~ydlu~ero~ide, 2-bu~dllone peroxide, ~o;~ tor.., e.g. azodiisobutylnitrile and 25 ~7,n~;r~-1,on~,--de, riboflavin + visible light, methylene blue + a redox couple, and the 1ike. Pr~,f~.~ly a ~h~m;c~l polymerization initiator such as pe. tul~ha~e will be employed. When necç ,,j~ . y to limit exposure of the monomers to oxygen during poly..,e,i~aLion, pol~",~ ion may be carried out in an oxygen free atmosphere, such a n i~lc.~ l al",o;",he.e.
Following poly"l~;-i~ion, the resultant copolymers in co-"bi.. dLion with a fluid phase can be used directly as a separation n.edi~ l for dcoL.o~uhoresis, where the concf~ Lion of the copolymers in the fluid phase provides for a hydrogel having WO 97/26288 PCTtUS97/00411 - 1 - u~ t~es suitable for use in eleu~ opholeD;s, or the copolymers can be sel,a- ~L~,d from the fluid phase and stored until later use, as appropliale. The copolymers can be recovered from the fluid phase using any convenient means, such as freeze drying or To prepare thermoreversible hydrogels from the subject copolymers, a sufficient amount of copolymer will be co~ ;..ed with an aqueous me~i-lm, where the aqueousprovides the continllQus fluid phase ofthe hydrogel. Generally, the amount of copol~ f that is co...l.i~fd with the a~ eouC ."~1;..,., will range from about 1 to 30 %T, and will usually range from about 2 to 20 %T, more usually from about 3 to 15 lû %T, where %T refers to the total weight of copolymer in grams per 100 ml of aqueous meriillm As described above, the aqueous mP~ m may comprise various agents that find use in elc~,~, uphoi e.,is, such as l>u~. ing salts, denaturing agents, and the like.
The subject th~ ,ol~,.tcrsible hydrogels are cl-~cle-~ed by undergoing a D~bD~ physical change over a narrow melting le...~ u-e range (Tm)~ where below 15 the Tm the hydrogel is present as a gel like composition having a high viscosity and capable of ele~,~-ophoretic sieving. Above the Tm of the hydrogel, the hydrogel is present as a flowable, pourable cu...poD;lion having a low viscosity and being h~capablc of ele~,L~upho.e~ic sieving. The Tm of a particular hydrogel accc.dil-g to the subject invention, as well as the physical p. upe~ Lies of the hydrogel above and below the Tm~
will depend on the both the nature of the particular copolyrner from which the gel is p-~ d, as well as the concel.L.~lion of the copolymer in the aqueous phase.
Generally, the subject hydrogels will have a Tm between about 5 and 80~C, usually bt:t~.ecn about 10 and 70 ~C, and more usually between about 15 and 65 ~C. The m~gnit~lde ofthe narrow range ofthe Tm will range from about 0.1 to 10 ~C, and will usually range from a-hout 0.1 to 7.5 ~C, more usually from ahout 0.1 to 5.0~C. Above the Tm~ the subject hydrogels will have a ~risco:,;ly ranging from about 5 to 30,000 cps, more usually from about 20 to 10,000 cps, while below the Tm the subject l"rdlog~,ls will have a sllhst~nti~lly infinite viscosity.
dition to the subiect copolymers described above, the the, ~l~u~ ersible hydrogels may further comprise one or more additional, non-p.ot~ ceQ~Is~ polymers that serve to mo(~ te the physical and/or sieving characteristics of the hydrogel. These ~clrlitic~nS~I polyrners may be linear or b-anclled, and include hydroxyethylc~ llos~
- ily~lluAy~ yyl cpllnlos~ methyl cf llulosç, llydlu~y~JIo~Jyl methyl cfAIIt~lose, polyethylene glycol, polyethylene oxide, galacto ..~I~nAll pullulan, dextran, polyvinyl alcohol, agarose, polyacryloylamino-etho~y~;lhanol and the like The weight pw~,e,-lage of these arl~tition~l polymers present in the hydrogel per milltlifPr of a~ueous phase will 5 depend on the particular ~ ition~l poly",er, the copolymer and the desired chara~ lics of the hydrogel. Generally, the T of the additional polymer or polymers in the hydrogel will range from 0 1 to 5.0 %, more usually from 0 1 to 2.0 %
The subject ll~.",u~ rsi~le Lydlogf ls find use as separation media for ele~l-ùphoretic appli~ ~;on~ Elecllophol~t;c a~plic~l;Qn~ in which the subject 10 hydrogels find use as separation media are well known, being des~i,il,ed in Andrews, Ele~ ophu~ s (1986) and Barron & Blanch, Separation and Purification Methods (1995) 24:1-118, and need not be reviewed in great detail here Briefly, in e le~,l.ophu-f .ic appli--,.t;onc, the subject media will be placed in an electrophoretic separation chal.~ , e.g a slab gel CO~ f l, colurnn, t h~nnçl, capillary and the like, of 15 an ele~l,ùpho~Lic device Although the cleclrophoresis device can be pl~,tJâl~,;l by producing the the"nol~ ersible hydrogel in situ, typically a pre-p,e,oa,Gd hydrogel will be introduced into the ele~ ùphGl ~lic ~hallll)el ~ where the hydrogel is in the first, flowable state Thus, the hydrogel may be pr-_p&r~d as described above and then introduced into the separation ~h~mbPr of the electrophoretic device when the 20 te"~p."al.lre of the hydrogel is above the Tm~ i.e. as a pregel solution. After being introduced into the ;,cpa, ~lion .,h~,.l.er, the te...~ re of the hydrogel may then be lowered so that the hydrogel ~nm~os a gel state, capab}e of elccl,ophoretic sieving The hydrogel may be introduced into the separation chan,be, using any convenientmeans Thus, for slab gel holders, it may be s~;cient to simply pour the pregel solution 25 into the s1ab gel holder while the hydrogel is above the Tm. Alternatively, for capillary holders, it may be more convenient to introduce the gel, while in a fluid state, into the interior volume of the capillary through inje~,lion or suction.
Once the hydrogel has been introduced into the separation çh~mh~r of the elecll ~,phur~,lic device and the lt~ pe~lufe of the hydrogel reduced to below the Tm~ a 3Q sample may be introduced into the hydrogel for ele-,l,opho,~,i,;s Where convenient, the l,~d,ogel may be prc-ele~,Ln)phoresed, where pre-electrophoresis can serve a variet,v of ~U11303eS, such as for introduction of se~JalaLion buffer, and the like Sample -- components which may be separated in the subject hydrogels include nucleic acids, IJlOt,~,;lls, carbohydrates and the like. The sample may be introduced into the gel using a variety of methods, with the particular method sPlected dependent on the type of device being e.l~loyed. Electrophoresis of the sample in the hydrogel may then be carried out S in accol~ ce with known procedures.
Following ~lecl.ophGlc,~,;s, the separated sample components may be analyzed in the gel, e.g by staining and the like. The ele.,~ horetically se~ala~ed sample cc,.llpon~;llL~. may be ~-,.lloved from the gel for further analysis. Depen1i.lg on the part;cular ele~l-opholc~lic device being e.~loyed, separation may be accGlll~lished by 10 blotting, or by raising the tellli)e.aLu.~ ofthe hydrogel above the Tm and then extracting the sample co...pol1~,..L of the interest from the resultant fluid me~ m In addition to their use in cle.,~lupholes.s, the subject copolymers find use inother separation applications such as ~ ollla~ography, as well as in lllelllbl ~nes, controlled release compocitiorlc~ contact lenses, and the like.
The following PY~mples are offered by way of illustration and not by way of limit~fi- n.
~;.X~ K~ AT.
A number of dirrel ~ copolymers of acrylylglycin~m;~e and acrylamide were p. ~ ,d and their characteristics were colll~ared to homopolymers of polyacrylylgly~ le 25 A. Gels of Acrylylglycin~ e and Acrylamide Copolymers ~AGA) 1. T8% Copolymer AGA50 A copolymer of acrylyl~,ly~ ,.... de (AG) and acrylamide (AA) was plepaled by collllfilllng AG and AA mono.nGl~ in pure water at room tel.lp~,.al-lre with ~lln OlU~
pers~llph~te (APS) and N,N,N'N'-te~.a..lcLl.ylethylçneAi~mine (TEMED) as 30 polylll.,.i~aliOn illiLiaLOl :,. The weight percent ratio of AG to AA monomers was 50:~0.
The conf~ .aLion oftotal monomer prior to pol~llle~i~aLion was 8 %. Upon - polylllc. i~,aliOl~ at room ten~pGl ~ re, a solution of a linear viscous polymer was ol~lailled.
2 T7.3% Copolymer AGA 73 The procedure used to produce the T8 AGA50 was employed, except that the ratio weight percent of AG to AA was Ghànged to 73 :27, and the conce.,ll i.lion of total nolne. prior to poly.,~ ;alion was 7.3 %. Following poly...~ ion at room tc.ll~e.~L~ t a Il~G~ or~rcrsible clear gel was obtained. The resultant hydrogel had a T~"
of 36.5 ~C.
3. T6.4% Copolymer AGA90 The same procedure as used to prepare the T7.3%AGA73 gel was employed, except that the weight % ratio of AG to AA was rh~nged to 90:10 and the COllC~ lLlalion of total ",onol~er prior to polymerization was 6.4%. Poly",~ Lion yielded a l~ pu~,-,L gel at room te.ll~ lulG which had a Tm of 63.9 ~C. The resultant T6.4% AGA90 gel was observed to be m~ch~r~je~lly stronger and more stable than the T7.3% AGA73 gel.
4 T7% AGA72 Gel The same procedu, e used to prepare the above hydrogel compositions of e~ s 1-3 was employed, except that the weight % ratio of AG to AA was ~h~n~f~d to 72:28, and the concGllLl aLion of total monom~or prior to POlY~llGl i~aLion was 7 %
Following polylll~ili~Lion, a clear gel was obtained.
To study the sllit~bility of the resultant hydrogel as a separation m~ lm for ele~,L~ ophoresis, 0.5 rnl of 10 x TBE buffer and 0.1 ~11 of ethi~illm bromide were added to 10 rnl ofthe hydrogel pregel solution sr~ tiol at 80 ~C, to achieve a final TBE
cone~ lion of 0.5x. The resultant hydrogel was cooled in a refrigerator prior to use ;n clc~ ,pi-oresis. Separation of a 100 bp ladder and ~PX174/Hae III was carried out at 12 V/cm. The gel sepal~Lad 7 bands of a POS~;IJIG 15 bands of the 100 bp ladder and 6 bands of a possible 11 bands ofthe ~X174/Hae III.
Following polylll~ili~Lion, a clear gel was obtained.
To study the sllit~bility of the resultant hydrogel as a separation m~ lm for ele~,L~ ophoresis, 0.5 rnl of 10 x TBE buffer and 0.1 ~11 of ethi~illm bromide were added to 10 rnl ofthe hydrogel pregel solution sr~ tiol at 80 ~C, to achieve a final TBE
cone~ lion of 0.5x. The resultant hydrogel was cooled in a refrigerator prior to use ;n clc~ ,pi-oresis. Separation of a 100 bp ladder and ~PX174/Hae III was carried out at 12 V/cm. The gel sepal~Lad 7 bands of a POS~;IJIG 15 bands of the 100 bp ladder and 6 bands of a possible 11 bands ofthe ~X174/Hae III.
- 5. T5.3% AGA89 The same procedure used to prepare the above hydrogel compositions of 1-4 was employed, except that the weight % ratio of AG to AA was G~ 'gt~d to 89: 11, and the col~re .n alioll of total ~..o.~ prior to polylllc~i~aLion was 5.3 %. Following S pol~ ion, a hydrogel having a Tm of 57 ~C was oblailled which was ~l,unge. than the T7%AGA90 hydrogel.
The separation ~p~bility ofthe le~u~ hydrogel was studied using the same p~ucedu~e as that used in 4, above. The T5.3%AGA89 gel provided better separation of dsDNA than did the T7%AGA90 hydrogel de~.v, il,cd in 4, above.
The separation ~p~bility ofthe le~u~ hydrogel was studied using the same p~ucedu~e as that used in 4, above. The T5.3%AGA89 gel provided better separation of dsDNA than did the T7%AGA90 hydrogel de~.v, il,cd in 4, above.
6. T2.8% AGA 89.
Sllffi.~ient water was added to the T5.3 %AGA89 hydrogel to reduce the AGA89 conce"l,d~ion from 5.3% to 2.8%. The resultant composition provided a clear gel at room t.,."~.~,.alllre.
Sllffi.~ient water was added to the T5.3 %AGA89 hydrogel to reduce the AGA89 conce"l,d~ion from 5.3% to 2.8%. The resultant composition provided a clear gel at room t.,."~.~,.alllre.
7. T 5 3 %AGA 89 7. lM Urea Gel Sl-ffi~ nt urea was added to the composition of T5 3%AGA89 hydrogel at 85 ~C to achieve a urea conce-,l-aLion in the gel of 7. l M. Upon cooling of the hydrogel to room tc.--pe.dL-lre, a clear gel was ol~l~ined.
B. Gels of Polyacrylyl~ly~ ".;de Homopolymers (PAG) 1. T5% Homopolyrner PAG
10 ,ul TEMED, 20 ~1 10% APS and sufr,ci.,..l AG were added to 10 rnl pure 25 water to achieve an AG monomer conc~.,LIalion of 5%. Following pol~,c.i~dlion at room Iv~ e. alure, an opaque gel was ol~Lai.-ed that became clear when the Le-l.~.,.
ofthe gel was raised to 85 ~C. The resultant gel was not therrnoreversible.
2. T5 5% PAG
0.5 g AG, 9g H20, 0.2 g isop.u~.yl alcohol, 20 1ll TEMED and 40 ~11 10% APS
were cc,ml,i-led at room Le -Il~c;- aL~ . Following pol~,.,t;.; aLiOn~ a gel was ob~di.-ed that was opa~ue at 25 ~C and clear at 90 ~C. The resultant gel was not tllel-,,vrc~rersible.
WO 97126288 PCT/~JS97/00411 -- 3. T 5.3%PAG.
This gel was p.c,oa, ,d in the manner as the gel in 2, except that polyme.~a~iol~
was carried out 65 ~C. Upon poly--~ aLion, a reversible gel was obtained with a Tm of 71 ~C
C. Comparison of AGA Hydrogels to PAG Gels In co...pa,i-,g the p,~,pe,Lies ofthe AGA hydrogels to the gels of PAG
homopolymer, severa~ di~ Gllces become a~)are,ll. While gels of PAG polymerized in pure water have been ,epo,Led to form in~olll~'e, though water swellable gels, 10 hydrogels of AGA in pure water were found to gel at room tellly~ lul ~ and dissolve upon heating. Fu- lhc~no~ while small ~ ntifies of hydrogen bond breaking reagents such as urea and thiocyanate have been reported to readily dissolve PAG gels, the T5.3%AGA89 gel was found to be stable at urea conce,.l,ations in excess of 7M~
While the ~flitiQn of water to PAG gels has been lepo-led to dissolve the gels, it was 15 found that addition of a ci~lifir,Ant amount of water to the T5.3%AGA89 gel did not dissolve the gel. Finally, while a 5.27 % PAG gel has been reported to have a Tm of 24 ~C, the Tm of TS.3%AGA89 which has roughly the same concentration of polymer wasfound to be 57 ~C, which is ~ ificS~ntly higher.
I~ .ll ogcls comprising the AGA copolymer were found to have much higher 20 sll~ glh and elasticity than PAG homopolymer gels, malcing them con",alaLi~ely easier to m~nipul~te FUIl1I~IIIIGI~:~ hydrogels con-pli~;"g the AGA copolymer are Ll~l~al~,n~
and highly l~yd~oplfilic, and provide for e~cellçnt electrophoretic separation when the gels are employed as electrophoretic separation media.
From the above results and ~lic~ ;on~ it is appa,en~ that thermoreversible hydrogels particularly suited for use as separation media for clc~,llophoresis are provided. The th~ ,.o, ~.,rersible hydrogels are easy to prepare and use, are adaptable to a variety of ele~ opllorelic devices, buffer and denaturing systems, are . l ~ c~lly strong, are 1, a~ Jal ~.ll for easy sample idçntifir~tion, and are capable of providing for high resolution of separated sample co"",one.,~.
B. Gels of Polyacrylyl~ly~ ".;de Homopolymers (PAG) 1. T5% Homopolyrner PAG
10 ,ul TEMED, 20 ~1 10% APS and sufr,ci.,..l AG were added to 10 rnl pure 25 water to achieve an AG monomer conc~.,LIalion of 5%. Following pol~,c.i~dlion at room Iv~ e. alure, an opaque gel was ol~Lai.-ed that became clear when the Le-l.~.,.
ofthe gel was raised to 85 ~C. The resultant gel was not therrnoreversible.
2. T5 5% PAG
0.5 g AG, 9g H20, 0.2 g isop.u~.yl alcohol, 20 1ll TEMED and 40 ~11 10% APS
were cc,ml,i-led at room Le -Il~c;- aL~ . Following pol~,.,t;.; aLiOn~ a gel was ob~di.-ed that was opa~ue at 25 ~C and clear at 90 ~C. The resultant gel was not tllel-,,vrc~rersible.
WO 97126288 PCT/~JS97/00411 -- 3. T 5.3%PAG.
This gel was p.c,oa, ,d in the manner as the gel in 2, except that polyme.~a~iol~
was carried out 65 ~C. Upon poly--~ aLion, a reversible gel was obtained with a Tm of 71 ~C
C. Comparison of AGA Hydrogels to PAG Gels In co...pa,i-,g the p,~,pe,Lies ofthe AGA hydrogels to the gels of PAG
homopolymer, severa~ di~ Gllces become a~)are,ll. While gels of PAG polymerized in pure water have been ,epo,Led to form in~olll~'e, though water swellable gels, 10 hydrogels of AGA in pure water were found to gel at room tellly~ lul ~ and dissolve upon heating. Fu- lhc~no~ while small ~ ntifies of hydrogen bond breaking reagents such as urea and thiocyanate have been reported to readily dissolve PAG gels, the T5.3%AGA89 gel was found to be stable at urea conce,.l,ations in excess of 7M~
While the ~flitiQn of water to PAG gels has been lepo-led to dissolve the gels, it was 15 found that addition of a ci~lifir,Ant amount of water to the T5.3%AGA89 gel did not dissolve the gel. Finally, while a 5.27 % PAG gel has been reported to have a Tm of 24 ~C, the Tm of TS.3%AGA89 which has roughly the same concentration of polymer wasfound to be 57 ~C, which is ~ ificS~ntly higher.
I~ .ll ogcls comprising the AGA copolymer were found to have much higher 20 sll~ glh and elasticity than PAG homopolymer gels, malcing them con",alaLi~ely easier to m~nipul~te FUIl1I~IIIIGI~:~ hydrogels con-pli~;"g the AGA copolymer are Ll~l~al~,n~
and highly l~yd~oplfilic, and provide for e~cellçnt electrophoretic separation when the gels are employed as electrophoretic separation media.
From the above results and ~lic~ ;on~ it is appa,en~ that thermoreversible hydrogels particularly suited for use as separation media for clc~,llophoresis are provided. The th~ ,.o, ~.,rersible hydrogels are easy to prepare and use, are adaptable to a variety of ele~ opllorelic devices, buffer and denaturing systems, are . l ~ c~lly strong, are 1, a~ Jal ~.ll for easy sample idçntifir~tion, and are capable of providing for high resolution of separated sample co"",one.,~.
- All p~blic~l;QIl~ and patent app~ ;on~ mentioned in this spe~ific~tion are herein inco",orated by ~ ~rt;rence to the same extent as if each individual publication or patent application was specifically and individually inrii~ted to be inco.~o.aled by l~,f~ c~. , s The invention now being fully des~,-il.cd, it will be a~pa- .,.~1 to one of ol dinal y skill in the art that many ÇllAn~ and mod~r~ s can be made thereto without departing from the spirit or scope of the appended claims
Claims (24)
1. A non-ionic, linear copolymer capable of forming a thermoreversible hydrogel when combined with an aqueous phase, said copolymer comprising:
a polyacrylamide backbone, wherein 55:45 to 95:5 weight percent ratio of the acrylamide monomeric units of said copolymer comprise N-substituent groups capable of hydrogen bonding.
a polyacrylamide backbone, wherein 55:45 to 95:5 weight percent ratio of the acrylamide monomeric units of said copolymer comprise N-substituent groups capable of hydrogen bonding.
2. The copolymer according to Claim 1, wherein said copolymer has a molecular weight of at least about 10 kD.
3. The copolymer according to Claim 1, wherein said N-substituent groups capable of hydrogen bonding comprise a carbamyl group as a hydrogen bonding moiety.
4. The copolymer according to Claim 1, wherein said hydrogen bonding group is a heterocyclic nitrogen base.
5. The copolymer according to Claim 1, wherein said hydrogen bonding group is an .alpha.-aliphatic amide.
6. A non-ionic, linear copolymer capable of forming a thermoreversible hydrogel when combined with an aqueous phase, said copolymer comprising:
first and second monomeric units in a weight percent ratio of from about 55:45 to about 95:5, wherein said first monomeric unit is of the formula:
wherein:
X is H or CH3, Y is a bond or a linking group; and Z is a group comprising a hydrogen bonding moiety, wherein said hydrogen bondingmoiety is of the formula:
D
wherein:
A is C or a heteroatom;
D is O or S; and R1 is H or an aliphatic substituent of up to 10 carbon atoms; and said second monomeric unit is acrylamide.
first and second monomeric units in a weight percent ratio of from about 55:45 to about 95:5, wherein said first monomeric unit is of the formula:
wherein:
X is H or CH3, Y is a bond or a linking group; and Z is a group comprising a hydrogen bonding moiety, wherein said hydrogen bondingmoiety is of the formula:
D
wherein:
A is C or a heteroatom;
D is O or S; and R1 is H or an aliphatic substituent of up to 10 carbon atoms; and said second monomeric unit is acrylamide.
7. The copolymer according to Claim 6, wherein Z is a heterocyclic nitrogen base selected from a purine or pyrimidine.
8. The copolymer according to Claim 6, wherein Z is an .alpha.-aliphatic amide.
9. The copolymer according to Claim 6, wherein said linking group is aliphatic chain of from 1 to 6 carbon atoms.
10. A copolymer of acrylylglycinamide and acrylamide, wherein the weight percent ratio of acrylylglycinamide to acrylamide of said copolymer ranges from about 55:45 to about 95:5.
11. A thermoreversible hydrogel suitable for use as a separation medium for electrophoresis, said hydrogel comprising:
a non-ionic, linear copolymer comprising a polyacrylamide backbone, wherein 55:45 to 95:5 weight percent ratio of the acrylamide monomeric units of said copolymer comprise N-substituent groups capable of hydrogen bonding; and a continuous fluid phase.
a non-ionic, linear copolymer comprising a polyacrylamide backbone, wherein 55:45 to 95:5 weight percent ratio of the acrylamide monomeric units of said copolymer comprise N-substituent groups capable of hydrogen bonding; and a continuous fluid phase.
12. The thermoreversible hydrogel according to Claim 11, wherein said thermoreversible hydrogel has a Tm ranging from about 15 to 65 °C.
13. The thermoreversible hydrogel according to Claim 11, wherein the amount of said copolymer in said hydrogel ranges from about 1 to 30 %T.
14. A thermoreversible hydrogel suitable for use as a separation medium for electrophoresis, said hydrogel comprising:
a non-ionic, linear copolymer comprising first and second monomeric units in a weight percent ratio of from about 55:45 to about 95:5, wherein said first monomeric unit is wherein said first monomeric unit is of the formula:
wherein:
X is H or CH3, Y is a bond or a linking group; and Z is a group comprising a hydrogen bonding moiety, wherein said hydrogen bondingmoiety is of the formula:
wherein:
A is C or a heteroatom;
D is O or S; and R1 is H or an aliphatic substituent of up to 10 carbon atoms; and said second monomeric unit is acrylamide; and a continuous liquid phase.
a non-ionic, linear copolymer comprising first and second monomeric units in a weight percent ratio of from about 55:45 to about 95:5, wherein said first monomeric unit is wherein said first monomeric unit is of the formula:
wherein:
X is H or CH3, Y is a bond or a linking group; and Z is a group comprising a hydrogen bonding moiety, wherein said hydrogen bondingmoiety is of the formula:
wherein:
A is C or a heteroatom;
D is O or S; and R1 is H or an aliphatic substituent of up to 10 carbon atoms; and said second monomeric unit is acrylamide; and a continuous liquid phase.
15. The thermoreversible hydrogel according to Claim 14, wherein the amount of said copolymer in said hydrogel ranges from about 1 to 30 %T.
16. The thermoreversible hydrogel according to Claim 14, wherein said first monomeric unit is acrylylglycinamide.
17. A thermoreversible hydrogel suitable for use as a separation medium for electrophoresis, said hydrogel comprising:
a polyacrylamide backbone, wherein a portion of the acrylamide monomeric units of said copolymer comprise N-substituent groups capable of hydrogen bonding;
an additional, non-proteinaceous linear polymer; and an aqueous phase.
a polyacrylamide backbone, wherein a portion of the acrylamide monomeric units of said copolymer comprise N-substituent groups capable of hydrogen bonding;
an additional, non-proteinaceous linear polymer; and an aqueous phase.
18 The thermoreversible hydrogel according to Claim 17, wherein said acrylamide monomeric units comprising N-substituent groups capable of hydrogen bonding are acrylylglycinamide.
19. A method of performing electrophoresis on a sample, the improvement comprising:
employing as a separation medium for said electrophoresis the thermoreversible hydrogel according to Claim 11.
employing as a separation medium for said electrophoresis the thermoreversible hydrogel according to Claim 11.
20. A method of performing electrophoresis on a sample, the improvement comprising:
employing as a separation medium for said electrophoresis the thermoreversible hydrogel according to Claim 14.
employing as a separation medium for said electrophoresis the thermoreversible hydrogel according to Claim 14.
21. The method according to Claim 20, wherein said first monomeric unit is acrylylglycinamide.
22. An electrophoretic device for performing electrophoresis on a sample, said device comprising:
an electrophoretic separation chamber comprising a thermoreversible hydrogel according to Claim 11.
an electrophoretic separation chamber comprising a thermoreversible hydrogel according to Claim 11.
23. The device according to Claim 22, wherein said device is a slab gel electrophoresis device.
24. The device according to Claim 22, wherein said device is a capillary electrophoresis device.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/589,026 | 1996-01-19 | ||
| US08/589,026 US5631337A (en) | 1996-01-19 | 1996-01-19 | Thermoreversible hydrogels comprising linear copolymers and their use in electrophoresis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2243601A1 true CA2243601A1 (en) | 1997-07-24 |
Family
ID=24356288
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| CA002243601A Abandoned CA2243601A1 (en) | 1996-01-19 | 1997-01-15 | Thermoreversible hydrogels comprising linear copolymers and their use in electrophoresis |
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| Country | Link |
|---|---|
| US (1) | US5631337A (en) |
| EP (1) | EP0874868A4 (en) |
| JP (1) | JP2000503698A (en) |
| AU (1) | AU712081B2 (en) |
| CA (1) | CA2243601A1 (en) |
| WO (1) | WO1997026288A1 (en) |
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| US9835587B2 (en) | 2014-04-01 | 2017-12-05 | C.C. Imex | Electrophoresis running tank assembly |
Family Cites Families (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3452182A (en) * | 1964-07-24 | 1969-06-24 | Polaroid Corp | Photographic products and processes |
| US4035319A (en) * | 1971-09-30 | 1977-07-12 | Polaroid Corporation | Non-migratory hardeners |
| US3762927A (en) * | 1971-09-30 | 1973-10-02 | Polaroid Corp | Silver halide emulsion containing a nonmigratory hardener |
| US3879205A (en) * | 1971-10-08 | 1975-04-22 | Polaroid Corp | Method of preparing photosensitive silver halide emulsions |
| US4172934A (en) * | 1978-02-08 | 1979-10-30 | Minnesota Mining And Manufacturing Company | Acrylic functional aminocarboxylic acids and derivatives as components of pressure sensitive adhesives |
| US4806434A (en) * | 1984-07-06 | 1989-02-21 | Fuji Photo Film Co., Ltd. | Medium for electrophoresis |
| GB8713854D0 (en) * | 1987-06-13 | 1987-07-15 | Kozulic B | Poly-n-acryloyl-tris(hydroxymethyl)aminomethane gels |
| DE3843194A1 (en) * | 1988-12-22 | 1990-07-12 | Hoechst Ag | AMPHIPHILE MONOMERS WITH MIXED-CHAIN STRUCTURE AND POLYMERS AND FILM FROM AT LEAST ONE MONOMOLECULAR LAYER THEREOF |
| US5274167A (en) * | 1989-01-26 | 1993-12-28 | Bayer Aktiengesellschaft | Polymeriable optically active (meth) acrylic acid derivatives |
| US5338428A (en) * | 1993-05-28 | 1994-08-16 | California Institute Of Technology | Poly(N-Acylalkylenimine) electrophoresis support media |
| US5883211A (en) * | 1996-01-19 | 1999-03-16 | Aclara Biosciences, Inc. | Thermoreversible hydrogels comprising linear copolymers and their use in electrophoresis |
-
1996
- 1996-01-19 US US08/589,026 patent/US5631337A/en not_active Expired - Lifetime
-
1997
- 1997-01-15 JP JP9526074A patent/JP2000503698A/en active Pending
- 1997-01-15 EP EP97901980A patent/EP0874868A4/en not_active Withdrawn
- 1997-01-15 AU AU15759/97A patent/AU712081B2/en not_active Ceased
- 1997-01-15 CA CA002243601A patent/CA2243601A1/en not_active Abandoned
- 1997-01-15 WO PCT/US1997/000411 patent/WO1997026288A1/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| WO1997026288A1 (en) | 1997-07-24 |
| US5631337A (en) | 1997-05-20 |
| EP0874868A4 (en) | 2000-07-12 |
| AU712081B2 (en) | 1999-10-28 |
| EP0874868A1 (en) | 1998-11-04 |
| AU1575997A (en) | 1997-08-11 |
| JP2000503698A (en) | 2000-03-28 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |