Cellulose acetate electrophoresis is a type of zone electrophoresis that separates molecules in liquid form such as proteins on a membrane or stripe made of cellulose acetate. Developed by a German chemist, Joachim Kohn (1912-1987) in 1957, cellulose acetate electrophoresis was the second form of zone electrophoresis, after filter paper electrophoresis. It was also one of the earliest electrophoretic techniques adopted in clinical laboratories for routine diagnosis that is still in use to date (Rocco, 2005).
Principles of Cellulose Acetate Electrophoresis
In cellulose acetate electrophoresis, a membrane or stripe made of cellulose acetate is used as a support matrix to separate components in the sample. As with typical zone electrophoresis, electrophoretic separation occurs in a homogeneous buffer system. The support matrix or medium is submerged in an electrophoresis running buffer maintained at a definite pH value, and electrophoretic separation is conducted for a definite time (Westermeier, et al., 2005).
It can provide a molecular sieving effect on electrophoretic separation of the sample, based on the size of the pores between the molecules of the support matrix. It can also reduce convection currents that are generated from excessive heat, which occurs during electrophoresis. When the separation is completed, components of the sample are isolated into distinct bands or zones. Each band represents constituents in the samples that possess similar or identical characteristics (Jorgenson, 1986; Walker, 2010).
In the case of cellulose acetate electrophoresis, membranes in the form of sheets or stripes made from cellulose acetate are saturated with the running buffer. Each end of the buffer-saturated stripes overlaps with a filter paper wick that reaches into the buffer in an electrophoresis tank (Figure 1). Samples of interest are dotted onto the surface at about half or one-third of the length of the cellulose acetate membrane (Kohn, 1969; Walker, 2010). The separation begins when an electric current is applied to each end of the stripe, which acts as an anode and cathode. At the end of the electrophoresis, separated components can be stained and unstained for visualization purposes and for further quantitative analyses (Kohn, 1962).
Cellulose acetate electrophoresis is suitable for rapid screening purposes by comparing the pattern of separation obtained from the sample of interest with that of a known reference. The technique is applicable to various types of samples, such as nucleic acids, proteins, polypeptides dyes and mixtures of dyes and polysaccharides (Kohn, 1962; Dudman & Bishop, 1968).
Cellulose Acetate as Electrophoretic Support Matrix
Cellulose acetate is derived from the acetylation of filter papers, which are made of pure cellulose. The acetylation is generally in the C-3 and C-6 positions of the glucose ring (Rochetti & Gelfi, 2001). Cellulose acetate possesses larger pores than other popular electrophoretic matrices such as agarose and polyacrylamide (Walker, 2010). The sieving influence that cellulose acetate imposes onto the separating components, therefore, are very little or no. In fact, it can be inferred that the mobility of the components in the sample during cellulose acetate electrophoresis is largely based on the overall charge rather than the size of the component in question (Kohn, 1957; Westermeier, et al., 2005).
For this reason, the ability to distinguish different components that are very similar in size, or the resolution of a cellulose acetate electrophoresis, is rather limited, when compared to gel or capillary electrophoresis. By the same token, cellulose acetate electrophoresis, unlike other matrices, is applicable for the separation of peptides or proteins based on their isoelectric point (pI)—where the overall charge of the peptides or proteins is zero. Electrophoresis of proteins or peptides on cellulose acetate supplies similar results to those obtained from isoelectric focusing (IEF) although cellulose acetate electrophoresis is not conducted in a pH gradient.
Advantages of Cellulose Acetate Electrophoresis
Cellulose acetate electrophoresis was first introduced when Kohn demonstrated its ability to separate hemoglobin, a protein in the red blood cell, and to identify abnormal hemoglobin in blood serum (Kohn, 1957 as cited in Rocco, 2005). The technique was very well-received among clinical biochemists because cellulose acetate provides several advantages over filter paper. The followings highlight some of the advantages of the technique:
1. Electrophoretic separation requires shorter time than filter paper electrophoresis
Filter paper electrophoresis, the predecessor of cellulose acetate electrophore