Although one of the older methods, cellulose acetate electrophoresis still has a number of applications. In particular, it has retained a use in the clinical analysis of serum samples, but is also used in laminar flow devices. Cellulose acetate has the advantage over paper in that it is a much more homogeneous medium, with uniform pore size, and does not adsorb proteins in the way that paper does. There is therefore much less trailing of protein bands and resolution is better, although nothing like as good as that achieved with polyacrylamide gels. The method is, however, far simpler to set up and run. Single samples are normally run on cellulose acetate strips (2.5 cm × 12 cm), although multiple samples are frequently run on wider sheets. The cellulose acetate is first wetted in electrophoresis buffer (pH 8.6 for serum samples) and the sample (1–2 mm³ ) loaded as a 1 cm wide strip about one-third of the way along the strip. The ends of the strip make contact with the electrophoresis buffer tanks via a filter paper wick that overlaps the end of the cellulose acetate strip, and electrophoresis is conducted at 6–8 V cm⁻¹ for about 3 h. Following electrophoresis, the strip is stained for protein, destained, and the bands visualised. A typical serum protein separation shows about six major bands. However, in many disease states, this serum protein profile changes and a clinician can obtain information concerning the disease state of a patient from the altered pattern. Although still frequently used for serum analysis, electrophoresis on cellulose acetate is being replaced by the use of agarose gels, which give similar, but somewhat better, resolution. A typical example of the analysis of serum on an agarose gel is shown in Figure 1. Similar patterns are obtained when cellulose acetate is used.

Fig1. Electrophoresis of human serum samples on an agarose gel. Tracks 2, 4, 5 and 6 show normal serum protein profiles. Tracks 1, 3 and 7 show myeloma patients, who are identified by the excessive production of a particular monoclonal antibody seen in the IgG fraction. (Courtesy of Charles Andrews and Nicholas Cundy, Edgware General Hospital, London.)

Enzymes can easily be detected in samples subjected to electrophoresis on cellulose acetate, by using the zymogram technique. The cellulose strip is laid on a strip of filter paper soaked in buffer and substrate. After an appropriate incubation period, the strips are peeled apart and the paper zymogram treated accordingly to detect enzyme product; hence, it is possible to identify the position of the enzyme activity on the original strip. An alternative approach to detecting and semi-quantifying any particular protein on a strip is to treat the strip as the equivalent of a protein blot and to probe for the given protein using primary antibody and then enzyme-linked secondary antibody. Substrate colour development indicates the presence of the particular protein and the amount of colour developed in a given time is a semiquantitative measure of the amount of protein. Thus, for example, large numbers of serum samples can be run on a wide sheet, the sheet probed using antibodies, and elevated levels of a particular protein identified in certain samples by increased levels of colour development.

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مواضيع ذات صلة

Protein (Western) Blotting

Detection, Estimation and Recovery of Proteins in Gels

Other Methods of Protein Purification

Chromatographic Methods for Protein Purification

Producing Recombinant Protein

Engineering Proteins for Purification

حوامض أمينية محورة Modified Amino Acids

Amplification methods

Immunoelectrophoresis

Immunoprecipitation

Antibody production

Introduction to Immunological methods