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Cellulose Acetate | Gel Electrophoresis | Molecular Biology

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  Table 1  Historicalsequenceof mainapplicationsof CA to elec-trophoretic protocols in different areas of research and clinicalinvestigations Year Application  1957 CA is used as an electrophoretic support (Kohn)1971 Application to conventional electrophoresis of white celland red cell enzymes (Meera Khan)1975 Application to isoelectric focusing of alpha-1-antitrypsinin human serum and 6 } phosphogluconate dehydro-genase (Harada)1984 Application to counterflow affinity isotachophoresis ofantigens in biological fluids with low protein contents(Abelev and Karamova)1992 Introduction of CA for protein transfer from polyacrylam-ide gels1993 Introduction of protocols for reusing CA zone sharpening make it possible to obtain, in par-ticular cases, much better results than when usingCZE. Most promising is the combination of ITP withCZE where ITP serves as a preconcentration andpre-separationstep for analysis of samples with com-plex matrices. Unfortunately, there is only onemanualITP } CZEsystemstillcommerciallyavailable. Further Reading Boc    \ ek P, Deml M, GebauerPand Doln m  H k V (1988) Analyti-cal Isotachophoresis , pp. 5 } 237. Weinheim: VCH.Boc    \ ek P, Gebauer P, Doln m  H k V and Foret F (1985) Recentdevelopments in isotachophoresis.  Journal of Chromatography  334: 157 } 195.Everaerts FM, Beckers JL and Verheggen ThPEM (1976) Isotachophoresis . Theory , InstrumentationandApplica-tions , Journal of Chromatography Library, vol. 6, pp.7 } 282. Amsterdam: Elsevier.Gebauer P and Boc    \ ek P (1997) Recent application anddevelopments of capillary isotachophoresis.  Elec-trophoresis  18: 2154 } 2161.Hirokawa T, Watanabe K, Yokota Y and Kiso Y (1993)Bidirectional isotachophoresis.  Journal of Chromatog-raphy  633: 251 } 259.Hjalmarsson SG and Baldesten A (1981) A critical reviewof capillary isotachophoresis.  CRC Critical Reviews inAnalytical Chemistry  11: 261 } 352.Kaniansky D and Mara  H k J (1990) On-line coupling of capillary isotachophoresis with capillary zone elec-trophoresis.  Journal of Chromatography 498: 191 } 204.Thormann W (1990) Isotachophoresis in open-tubularfused-silicacapillaries.Impactofelectroosmosisonzoneformation and displacement.  Journal of Chromatogra- phy  516: 211 } 217. Cellulose Acetate G. Destro-Bisol , University ‘La Sapienza’,Rome, Italy M. Dobosz and V. Pascali , Catholic University,Rome, ItalyCopyright  ^  2000 Academic Press The introduction of zone electrophoresis, pioneeredbyKonigin1939,playedacrucialroleintheprogressof electrokinetic separations. With this technique,molecules migrate as zones with sharp boundaries ina supporting medium immersed in a buffer solutionunder the application of an electric  R eld. Zone elec-trophoresis was quickly found to be superior in per-formance to Tiselius’s srcinal technique of movingboundaryelectrophoresis and replaced it entirely  }  tobesupersededinturnbydisplacementelectrophoresisand isoelectric focusing (IEF). Interestingly, the term‘zone electrophoresis’ was  R rst suggested by Tiseliushimself.Kohn  R rst used cellulose acetate (CA) as a support-ing medium for zone electrophoresisin 1957, as a su-perior substitute for plain  R lter paper. Since then, CAhas been used in many electrophoretic protocols, forboth research and clinical investigations ( Table 1 ).Nowadays CA electrophoresis is a widespreadtechnique.In this article we explain what CA is and why it isused in electrophoresis. This is followed by a brief overview of the uses of CA in various electrophoreticcontexts.Finally,somerecentandinnovativeapplica-tions of CA in electrophoretic protocols are dis-cussed. General Concepts Preparation of CA  CA sheets employed in electrophoresis are made of a molecular matrix, similar in structure to a spongebut a thousand times smaller. This matrix is obtainedby letting acetic anhydride react with cellulose anddissolving the product in an organic solvent, that canevaporate quickly. After letting the solvent evaporatein closely-controlled conditions of temperature andhumidity, a highly permeable matrix is obtained witha uniformly distributed microporosity. The spatialvolume of the pores may account for 80% of thetotal matrix size, ensuring ideal permeation by any 1222  II  /  ELECTROPHORESIS  /  Cellulose Acetate  electrolytic solution. When shapedinto gel sheets CAhas better resistance to the dehydration involved inthe dissipation of heat and is more easily handled.Thus, pre-gelled CA membranes (also referred to asCellogel  ) are the  R rst choice of support for manyelectrophoretic applications. For better handling,somecommercialversionsofCellogel TM comeweldedto an inert support of polyester plastic (Mylar  ).These commercial forms of CA pass practically un-changed through the entire separation } staining } de-staining cycle of a classical electrophoretic experi-ment.There are several major factors accounting for theversatile electrophoretic properties of CA: (1) thecellulose chain length, which ranges from a few tomillions of individual molecules; (2) the degree of acetylation (from 0.1% to 44%); (3) the pore size(between50 A       and10   m), therandomporedistribu-tion and the volume of the pores compared with thesolid matrix (20% to 80%). The spatial coiling of cellulose molecules, the type and concentration of wetting agents and the presence of residual con-taminants may also be important factors. CA as an Electrophoretic Medium Migration of molecules through the CA matrix de-pends mainly on the nett charge on the molecule, thebuffer pH and ionic strength and the intensity of theelectric  R eld. The difference in surface nett chargebetween the molecular species in a sample to beseparated is perhaps the most important point toconsider. Proteins are amphoteric, like their constitu-ent amino acids, and they may be charged positivelyor negatively depending on the pH of the solventmedium (the buffer solution, in an electrophoreticexperiment).Ingel electrophoresisa sievingeffect may affect theseparation, depending on the critical relationship be-tween the spatial shape of a protein species and thepore size of the matrix medium. Because of the ex-tremely large cellulose matrix pores, the mobility of proteins in CA electrophoresis is a direct function of their surface net charge, whereas molecular weightand shape are less important. For example, the hu-man heavy   -2 macroglobulin ( M r : 1000000; p I    5.9)moves faster than the much lighter haptoglobin ( M r 100,000; p I    6.1) in alkaline buffer solutions.As in most electrophoretic protocols, to improvea CA separation the ideal buffer pH and ionicstrength, strip temperature, voltage, current, elec-troosmosisand time of separation shouldbe selected.The optimal ionic strength is between 0.01 and 0.1(mequivL  1 ). Although mobility is theoretically en-hanced at high temperature, proteins are easily heatdenatured so the separation temperatures should bekeptbelow50 3 C.Moreover,sinceCAelectrophoresisis traditionally carried out with no cooling, separ-ation voltages should not exceed 500 V (60 V perlinear centimetre in gel strips),and the current shouldbe adjusted to less than 2.5 mA cm  2 . CA containspolar groups  }  hydroxy (OH  ) and acetyl(CH 3 COO  ) radicals  }  that become charged at thepH system and move towards the anode through thecellulose matrix. This produces a counter-reaction,displacing buffer toward the cathode and interferingwith the separation of the molecules of interest (en-doosmosis). Prolonged separation times may thuslead to the creation of artefacts due to the combinedeffects of heat, buffer turbulence and the counter-diffusion of molecules. Running times should be al-tered accordingly.A few fundamental properties make CA elec-trophoresis notably superior to electrophoresis using R lter paper: (1) the CA matrix is homogeneous,microporous and chemically pure, reducing molecu-lar adsorption to a minimum; (2) instant heat dissi-pation occurs in the matrix, which does not need tobe cooled; (3) the amount of protein needed is verysmall (1 mg or less); (4) the inherent buffering } staining } destainingsystem is very simple; (5) stainedCA strips have no background;(6) the standard elec-trophoreticapparatusrequiredissimpleandinexpen-sive ( Figure 1 ).For most purposes  }  especially for routine clinicalinvestigations  }  small-scale CA electrophoresis (withmembranes ( 10 cm long) is widely used ( Figures 2 and  3 ). Larger scale membranes (usually 20 cm long)suit a variety of research analytical purposes andmicropreparative applications. CA in Electrophoretic Protocols Conventional Electrophoresis CA was srcinally introduced as a classical supportfor analytical zone electrophoresis but found a muchbroaderrange of applications. Essentially, it can nowbe used for both analytical and preparative purposes.Preparative applications exploit the speed of CA sep-arations, the absence of molecular interaction, andthe easy recovery of biologically active substancesfrom the matrix.CA is popular in clinical laboratories in whichsome well-established routine analyses are per-formed, e.g. for haemoglobin, serum proteins, lipo-proteins and lactate dehydrogenase. Isoforms of many enzymes and proteins from different tissuescome out very clear-cut on CA  }  a fact that is (or hasbeen) of particular interest for anthropogenetic and II  /  ELECTROPHORESIS  /  Cellulose Acetate  1223  Figure 1  Description of a universal electrophoretic apparatus for CA electrophoresis(redrawn and modified from Kohn, 1957). TheCAstrips(11)aresupportedateachendbytheshoulderpieces(4)andwhentautarejustclearofthetopedgeofthecentrepartition(10).The top edge of this centre partition is formed as a row of pyramids (9) which support the strip should it tend to sag. When using longstrips, strip supports (6) may be fitted to the labyrinth partitions (7) that form the connections between the buffer compartments (5)and electrode compartments (8). Filter paper wicks (3) connect the CA strips to the buffer compartments. The internal sides of thetank are stepped all round (2) as an aid to buffer level checking. The lid (12) fits in a recess (1) moulded all round the tank. Figure 2  Electrophoretic separation of human haemoglobin variants A, C and S. Ponceau red staining was used to visualizehemoglobin bands, and the anode was on top. forensic purposes and for the biochemical character-ization and classi R cation of various pathogenicmicroorganisms such as  Leishmania  and  Trypano-soma  species.In addition to one-dimensional electrophoreticmethods, two-dimensionalCA electrophoretic proto-cols are also available. A summary of important ap-plications is given in  Table 2 . Detection and Quantitation Any protein stain can be used with CA, providedthatthe solution does not contain a cellulose solvent.Aqueous staining solutions are preferred to alcoholicones, since with the latter strips tend to shrink andcurl unless they are passed through an aqueous bath.Staining solutions for CA are less concentrated thanthose used in  R lter paper electrophoresis, and theycan be repeatedly used with no appreciable loss of sensitivity.A wide range of analytical applications can belisted with an impressive variety of fully compatiblestainingmethods, including Coomassieblue brilliant,Ponceau red, Nigrosin, Schiff, gold and silver stain,different types of immuno-staining, and many differ-ent types of enzyme speci R c staining. A 5% (w /  w)aqueoussolution of acetic acid is a universal washingsolution for reducing the background.The simplest way of evaluating the results is byvisual inspection of stained strips, which should becarried out against a strong light source to improvethe assessment of the separation pattern. 1224  II  /  ELECTROPHORESIS  /  Cellulose Acetate  Figure 3  Routine clinical electrophoretic separations on CA:(A) serum proteins; (B) lipoproteins; (C) Lactate dehydrogenaseisoenzymes. Samples were obtained from healthy patients. Table 2  Some recent applications of CA electrophoresis Year Application  1994 Introduction of thermocooling apparatus for CA IEFSequential electrophoresis, with detection of 21 differentalleles in ESD-2 locus in  Drosophila buzzatii  1995 Improved separationof apolipoproteinsby use of surfac-tant Tween 201996 Rapid screening of biochemical loci of ratHighly sensitive detection of urinary proteins using col-loidal silver staining1997 Detection of superoxide dismutase isozymes to distin-guish between  tsetse   blood meals of human and non-human srcinCA electrophoresis used as the method of choice foralpha-thalassaemia screeningIEF on CA applied to the analysis of microheterogeneityof immunoglobulins and serum protein fraction Quantitative determinations can be carried out byelution or by scanning of the stained strips. Oncestained, protein bands can be easily eluted from themembrane by an appropriate buffer system (a classi-cal system is Tris (2-amino-2-hydroxymethyl-propane-1,3 } diol) or Barbitone elution buffer overPonceau red stained bands). Alternatively, a solvent(e.g. chloroform } ethanol 9:1 v /  v) can be used todissolve the membrane and recover the proteinof interest. To enhance the recovery ef  R ciency, gelledCA blocks (about 0.5 cm thick, instead of much thin-ner 0.5 mm supports) can be used.Scanning is preferred to elution for routine clinicalapplications.To reducebackgroundandincreasesen-sitivity,CAstripsshouldbeclearedpriortoscanning.As with  R lter paper it is important to use oil with thesame refractive index as the support. CA stripscleared with oil may be returned to their srcinal drystate by using a solvent such as ether. By contrast,swellingagents suchas acetic acid and dioxanusedinconjunction with heat treatment, permanently clearCA. Isoelectric Focusing CA has ideal features to suit IEF separations. CA isvirtually a non-sieving matrix enabling a quasi-freefractionation of macromolecules according to theirrespective isoelectric points (p I  , the pH at whichthereoccursanequalnumberofnegativeandpositivesurface charges). CA is easily soaked with very smallamounts of carrier ampholyte species, allowing themto be eluted in due course with no damage tostained } destained proteins; this in turn allows den-sitometry measurements and storage.Unfortunately, the combined effect of CA elec-troosmotic  S ow and the low ionic strength of com-mercial ampholinescan seriously impair the resultingseparation of proteins at their isoelectric points. Toovercome these drawbacks, CA has been variouslytreatedwithsurfaceactive agentsorwithmethylatingagents. Such treatments can partly  }  if not wholly }  reduce the osmotic  S ow. Also, a high concentrationof carrier ampholytes should be used to cover broadpH ranges (8% v /  v instead of the customary 2% v /  v)and electrolyte additives at low concentration (suchas 0.2 M lysine and 0.2 M acetic acid) should helpstabilize narrow pH intervals. Untreated CA stripsgive better results when 5%   -mercaptoethanol and5 M urea are used as stabilizing agents.Alternative strategies to circumvent electroosmo-sis, which differ in effectiveness, involve shorteningthe inter-electrode distance or using ‘chemical space-rs’ to  S atten the pH gradients at the appropriatesegment of separation. These devices may help tocreate high  R eld strengths with low voltages. Re-cently, thermoelectric cooling has been usedto stabil-ize CA IEF gradients. Counter ] ow Af \ nity Isotachophoresis Isotachophoresis or ‘displacement’ electrophoresispermitssimultaneousconcentrationandeffectivesep-aration of surface-charged substances, including bio-logical macromolecules. With this analytical method, II  /  ELECTROPHORESIS  /  Cellulose Acetate  1225
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