Cellulose Acetate

electrolytic solution. When shaped into gel sheets CA has better resistance to the dehydration involved in the dissipation of heat and is more easily handled. Thus, pre-gelled CA membranes (also referred to as Cellogel™) are the first choice of support for many electrophoretic applications. For better handling, some commercial versions of Cellogel™ come welded to an inert support of polyester plastic (Mylar™). These commercial forms of CA pass practically unchanged through the entire separation-staining-de-staining cycle of a classical electrophoretic experiment.

There are several major factors accounting for the versatile electrophoretic properties of CA: (1) the cellulose chain length, which ranges from a few to millions of individual molecules; (2) the degree of acetylation (from 0.1% to 44%); (3) the pore size (between 50 A and 10 |im), the random pore distribution and the volume of the pores compared with the solid matrix (20% to 80%). The spatial coiling of cellulose molecules, the type and concentration of wetting agents and the presence of residual contaminants may also be important factors.

CA as an Electrophoretic Medium

Migration of molecules through the CA matrix depends mainly on the nett charge on the molecule, the buffer pH and ionic strength and the intensity of the electric field. The difference in surface nett charge between the molecular species in a sample to be separated is perhaps the most important point to consider. Proteins are amphoteric, like their constituent amino acids, and they may be charged positively or negatively depending on the pH of the solvent medium (the buffer solution, in an electrophoretic experiment).

In gel electrophoresis a sieving effect may affect the separation, depending on the critical relationship between the spatial shape of a protein species and the pore size of the matrix medium. Because of the extremely large cellulose matrix pores, the mobility of proteins in CA electrophoresis is a direct function of their surface net charge, whereas molecular weight and shape are less important. For example, the human heavy a-2 macroglobulin (Mr: 1 000000; pI5.9) moves faster than the much lighter haptoglobin (Mr 100,000; pI6.1) in alkaline buffer solutions.

As in most electrophoretic protocols, to improve a CA separation the ideal buffer pH and ionic strength, strip temperature, voltage, current, elec-troosmosis and time of separation should be selected. The optimal ionic strength is between 0.01 and 0.1 (mequivL"1). Although mobility is theoretically enhanced at high temperature, proteins are easily heat denatured so the separation temperatures should be kept below 50°C. Moreover, since CA electrophoresis is traditionally carried out with no cooling, separation voltages should not exceed 500 V (60 V per linear centimetre in gel strips), and the current should be adjusted to less than 2.5 mAcm"2. CA contains polar groups - hydroxy (OH-) and acetyl (CH3COO) radicals - that become charged at the pH system and move towards the anode through the cellulose matrix. This produces a counter-reaction, displacing buffer toward the cathode and interfering with the separation of the molecules of interest (en-doosmosis). Prolonged separation times may thus lead to the creation of artefacts due to the combined effects of heat, buffer turbulence and the counter-diffusion of molecules. Running times should be altered accordingly.

A few fundamental properties make CA elec-trophoresis notably superior to electrophoresis using filter paper: (1) the CA matrix is homogeneous, microporous and chemically pure, reducing molecular adsorption to a minimum; (2) instant heat dissipation occurs in the matrix, which does not need to be cooled; (3) the amount of protein needed is very small (1 mg or less); (4) the inherent buffering-staining-destaining system is very simple; (5) stained CA strips have no background; (6) the standard elec-trophoretic apparatus required is simple and inexpensive (Figure 1).

For most purposes - especially for routine clinical investigations - small-scale CA electrophoresis (with membranes < 10 cm long) is widely used (Figures 2 and 3). Larger scale membranes (usually 20 cm long) suit a variety of research analytical purposes and micropreparative applications.

CA in Electrophoretic Protocols

Conventional Electrophoresis

CA was originally introduced as a classical support for analytical zone electrophoresis but found a much broader range of applications. Essentially, it can now be used for both analytical and preparative purposes. Preparative applications exploit the speed of CA separations, the absence of molecular interaction, and the easy recovery of biologically active substances from the matrix.

CA is popular in clinical laboratories in which some well-established routine analyses are performed, e.g. for haemoglobin, serum proteins, lipo-proteins and lactate dehydrogenase. Isoforms of many enzymes and proteins from different tissues come out very clear-cut on CA - a fact that is (or has been) of particular interest for anthropogenetic and

Figure 1 Description of a universal electrophoretic apparatus for CA electrophoresis (redrawn and modified from Kohn, 1957). The CA strips (11) are supported at each end by the shoulder pieces (4) and when taut are just clear of the top edge of the centre partition (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 long strips, 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 the tank 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 1 Description of a universal electrophoretic apparatus for CA electrophoresis (redrawn and modified from Kohn, 1957). The CA strips (11) are supported at each end by the shoulder pieces (4) and when taut are just clear of the top edge of the centre partition (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 long strips, 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 the tank 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.

forensic purposes and for the biochemical characterization and classification of various pathogenic microorganisms such as Leishmania and Trypanosoma species.

In addition to one-dimensional electrophoretic methods, two-dimensional CA electrophoretic protocols are also available. A summary of important applications is given in Table 2.

Detection and Quantitation

Any protein stain can be used with CA, provided that the solution does not contain a cellulose solvent. Aqueous staining solutions are preferred to alcoholic ones, since with the latter strips tend to shrink and curl unless they are passed through an aqueous bath.

Staining solutions for CA are less concentrated than those used in filter paper electrophoresis, and they can be repeatedly used with no appreciable loss of sensitivity.

A wide range of analytical applications can be listed with an impressive variety of fully compatible staining methods, including Coomassie blue brilliant, Ponceau red, Nigrosin, Schiff, gold and silver stain, different types of immuno-staining, and many different types of enzyme specific staining. A 5% (w/w) aqueous solution of acetic acid is a universal washing solution for reducing the background.

The simplest way of evaluating the results is by visual inspection of stained strips, which should be carried out against a strong light source to improve the assessment of the separation pattern.

Figure 2 Electrophoretic separation of human haemoglobin variants A, C and S. Ponceau red staining was used to visualize hemoglobin bands, and the anode was on top.
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