Iii

Figure 3 Routine clinical electrophoretic separations on CA: (A) serum proteins; (B) lipoproteins; (C) Lactate dehydrogenase isoenzymes. Samples were obtained from healthy patients.

Quantitative determinations can be carried out by elution or by scanning of the stained strips. Once stained, protein bands can be easily eluted from the membrane by an appropriate buffer system (a classical system is Tris (2-amino-2-hydroxymethyl-propane-1,3-diol) or Barbitone elution buffer over Ponceau red stained bands). Alternatively, a solvent (e.g. chloroform-ethanol 9: 1 v/v) can be used to dissolve the membrane and recover the protein

Table 2 Some recent applications of CA electrophoresis

Year Application

1994 Introduction of thermocooling apparatus for CA IEF Sequential electrophoresis, with detection of 21 different alleles in ESD-2 locus in Drosophila buzzatii

1995 Improved separation of apolipoproteins by use of surfac tant Tween 20

1996 Rapid screening of biochemical loci of rat

Highly sensitive detection of urinary proteins using colloidal silver staining

1997 Detection of superoxide dismutase isozymes to distin guish between tsetse blood meals of human and nonhuman origin

CA electrophoresis used as the method of choice for alpha-thalassaemia screening IEF on CA applied to the analysis of microheterogeneity of immunoglobulins and serum protein fraction of interest. To enhance the recovery efficiency, gelled CA blocks (about 0.5 cm thick, instead of much thinner 0.5 mm supports) can be used.

Scanning is preferred to elution for routine clinical applications. To reduce background and increase sensitivity, CA strips should be cleared prior to scanning. As with filter paper it is important to use oil with the same refractive index as the support. CA strips cleared with oil may be returned to their original dry state by using a solvent such as ether. By contrast, swelling agents such as acetic acid and dioxan used in conjunction with heat treatment, permanently clear CA.

Isoelectric Focusing

CA has ideal features to suit IEF separations. CA is virtually a non-sieving matrix enabling a quasi-free fractionation of macromolecules according to their respective isoelectric points (pi, the pH at which there occurs an equal number of negative and positive surface charges). CA is easily soaked with very small amounts of carrier ampholyte species, allowing them to be eluted in due course with no damage to stained-destained proteins; this in turn allows den-sitometry measurements and storage.

Unfortunately, the combined effect of CA elec-troosmotic flow and the low ionic strength of commercial ampholines can seriously impair the resulting separation of proteins at their isoelectric points. To overcome these drawbacks, CA has been variously treated with surface active agents or with methylating agents. Such treatments can partly - if not wholly - reduce the osmotic flow. Also, a high concentration of carrier ampholytes should be used to cover broad pH ranges (8% v/v instead of the customary 2% v/v) and electrolyte additives at low concentration (such as 0.2 M lysine and 0.2 M acetic acid) should help stabilize narrow pH intervals. Untreated CA strips give better results when 5% ^-mercaptoethanol and 5 M urea are used as stabilizing agents.

Alternative strategies to circumvent electroosmo-sis, which differ in effectiveness, involve shortening the inter-electrode distance or using 'chemical spacers' to flatten the pH gradients at the appropriate segment of separation. These devices may help to create high field strengths with low voltages. Recently, thermoelectric cooling has been used to stabilize CA IEF gradients.

Counterflow Affinity Isotachophoresis

Isotachophoresis or 'displacement' electrophoresis permits simultaneous concentration and effective separation of surface-charged substances, including biological macromolecules. With this analytical method, proteins are stacked as closely spaced, narrow bands between the 'leading' and the 'trailing' ions. Iso-tachophoresis on CA gels takes advantage of the absence of sieve effect in this matrix to study sets of interacting biological macromolecules, such as antigen/antibody and glycoprotein/lectin systems. However, electroosmosis once again interferes with this application. Abelev and Karamova were able to overcome this drawback by demonstrating that the cath-odic counterflow, combined with the constant flow of liquid through the membrane, stabilizes separations. The counterflow may be also used as a 'conveyer belt' to move immunoreagents through antigens or antibodies immobilized onto the membrane. Abelev and Karamova used a discontinuous buffer system, in which the two buffers have the same cation and differ in the anion species (chloride as the leading ion and ^-alanine as the trailing ion). Under these conditions, macromolecules are separated between the two anions.

Abelev and Karamova's method was originally developed to analyse proteins in highly dilute biological fluids such as urine, tears, and cerebrospinal and amniotic fluids, and it turned out to also be useful for detecting low levels of urinary monoclonal immunoglobulin light chains (Bence Jones protein) and alpha-fetoprotein in various pathological conditions.

CA as a Reusable Electrophoretic Support

CA separations are faster than those on other supports, usually with no resolution loss. However, CA sheets cost considerably more than starch, agar, agarose or polyacrylamide gel sheets.

Recently, a wash method has been described that makes it possible to recycle CA strips. The procedure has been shown to work even after using the strips for analysis of a variety of erythrocyte isoenzymes, which notoriously expose the support matrix not only to the strain of the electric field but also to many somewhat elaborate biochemical colorimetric treatment steps. Surprisingly, none of these stages seem to irreversibly affect the mechanical and physicochemical properties of the CA. In fact, after a variety of enzyme activity tests (adenosine deaminase, adenylate kinase, carbonic anhydrase, erythrocyte acid phosphatase, esterase D, glutathione peroxidase, glyoxalase 1, phosphog-lucomutase and 6-phosphogluconate dehydrogenase) Cellogelâ„¢ returns to its original features if soaked/ washed in water and methanol for a short time. In the course of double blind trials, no difference in band sharpness and resolution was noticed between new and used Cellogelâ„¢ strips. The procedure can be repeated two or three times if care is taken to avoid warping strips with absolute methanol soaking or rough handling.

Blotting Proteins from Polyacrylamide Gels to CA Sheets

Different electrophoretic species run in the same gel for the same time with the same electric field settings. The end of a given experiment is currently set depending on the specific requirements of the molecules to be separated, in zone electrophoresis as well as in IEF.

To achieve optimal resolution of different protein constituents of the same sample, various experiments are often carried out, only differing in voltage and duration. To save time, a simple method involves repeatedly blotting a polyacrylamide gel with CA sheets at various stages of separation. The blots obtained in this way can be stained and the protein species made to show the optimal resolution.

The advantages that can be obtained from CA blots of the same acrylamide gel are great, the most outstanding being:

1. various stages of a single protein separation can be tested in one experiment, to improve the protocol;

2. common and rare variants of a single elec-trophoretic pattern can be detected, each under optimal separation;

3. several proteins can be analysed at optimal conditions in the same experiment;

4. all the allele products may be discriminated by isotacophoretic mechanisms (in non-equilibrium IEF) and isoelectric point (in true equilibrium IEF) within the same run.

Conclusion

Almost uniquely among the various supports for elec-trokinetic separations, CA electrophoresis is still intensively used for both research and routine applications. The reasons for this long-lasting success are clear: simplicity of use, low cost, versatility and cost effectiveness. These same factors are likely to provide the general basis for the continuing use of CA in the future.

Acknowledgement

The drawing of Figure 1 was provided by Niccolo Falchi of the Department of Animal and Human Biology, University of Rome 'La Sapienza'.

Further Reading

Abelev GI and Karamova ER (1984) Counterflow affinity isotacophoresis on cellulose acetate membranes. Analytical Biochemistry 142: 437-444.

Ambler J (1978) Isoelectric focusing of proteins on cellulose acetate gel membranes. Clinica Chimica Acta 85: 183-191.

Destro-Bisol G and Santini SA (1995) Electrophoresis on cellulose acetate and Cellogel: current status and perspectives. Journal of Chromatography A 698: 33-40.

Golias TL (1971) Helena Laboratories Electrophoresis Manual. Beaumont, Texas: Helena Laboratories.

Grunbaum BJ, ed. (1980) Handbook for Forensic Individ-ualization ofHuman Blood and Bloodstains. Gottingen: Sartorius.

Harada H (1975) Isoelectrofocusing in cellulose acetate membrane: the method and application. Clinica Chimica Acta 63: 275-283.

Kohn J (1970) Electrophoresis and immunodiffusion techniques on cellulose acetate membrane. Methods in Medical Research 12: 243-260.

Meera Khan P (1971) Enzyme electrophoresis on cellulose acetate gel: zymogram patterns in man-mouse and man-Chinese hamster somatic cell hybrids. Archives of Biochemistry and Biophysics 145: 470-483.

Righetti PG (1976) Isoelectric Focusing, Theory, Methods and Applications. Amsterdam: Elsevier.

Schneider RG (1978) Methods for detection of hemoglobin variants and hemoglobinopathies in the routine clinical laboratory. CRC Critical Reviews in Clinical Laboratory Sciences 9: 243-271.

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