Cellulose Acetate Electrophoresis: Principle and Application

When analyzing different biochemical samples separating each component is an essential step. Electrophoresis is one of the methods of separation where elements of the sample are separated due to the influence of electric current. Electrophoresis has different types based on the supporting media used; polyacrylamide gel, agarose gel, capillary, and cellulose acetate electrophoresis. 

It is a simple method of separating components using a cellulose acetate strip as supporting media. Although it is an old separation method, it helps analyze serum, mainly hemoglobin, under alkaline conditions. The technique is easy to operate and less time-consuming.     

Principle of Cellulose Acetate Electrophoresis

The principle of this electrophoresis method is similar to the general principle of electrophoresis, where a support matrix (cellulose acetate strip) is used to separate sample components in a buffer system under electric volt.

The cellulose acetate is obtained from the acetylation of cellulose paper (filter paper). It is a suitable support matrix because it has larger pores than the agarose or polyacrylamide gels. After all, separation is not based on the size of the component. This property helps to analyze constituents of similar size easily using this technique. The separation of proteins and peptides in the cellulose acetate occurs based on the isoelectric point of the components, where the overall charge of proteins and peptides are zero. (1) 

The cellulose acetate strip is first wetted in the electrophoretic buffer. Then the sample is loaded in the strip (approximately ⅓ area of the strip). The end of the strip is in contact with the buffer with the help of a filter paper wick. The electric volt of 6-8 V is provided in the strip for about an hour. The components are separated in the form of a band based on their charge. (1) The positively charged substance moves to the cathode, and the negatively charged substance moves to the anode.

The separated band is detected using the zymogram method after applying current for the desired time. The strip is placed in filter paper, soaked in buffer and substrate, and incubated for one hour. The strips are peeled away from the filter paper; the zymogram paper is treated as per the required chemicals for suspected proteins. Other methods of semi-quantifying the separated band are treating it with an appropriate blotting technique or using a spectrophotometer.    

Materials Required for Cellulose Acetate Electrophoresis

1. Cellulose acetate strip/membrane: Cellulose acetate is a thermoplastic resin. The cellulose paper is acetylated by treating it with acetic anhydride. It is used as a support matrix. Here one side of the strip acts as the cathode and another as the anode.
2. Electrophoresis buffer: Tris-EDTA buffer (TEB) of 8.4 pH is usually used for analyzing blood. 
3. Electrophoresis tank: An horizontal electrophoresis tank with an adjustable bridge is used. 
4. Power supply: A power supply that can deliver constant current is required. It can go up to 400 volts. 
5. Filter paper wicks: 3mm Whatman filter paper wick with a length similar to the width of an electrophoretic tank. 
6. Spectrophotometer/zymogram paper/ materials for blotting techniques are required for detecting and quantifying the separated components. 

Application of Cellulose Acetate Electrophoresis

The most common application of cellulose acetate electrophoresis is in the diagnostic area for determining abnormalities in human hemoglobin.

So, it is sometimes referred to as hemoglobin electrophoresis. The applications of cellulose acetate electrophoresis are:

1. Blood analysis: The cellulose acetate electrophoresis is applicable to determine hemoglobin abnormalities, especially in people with undiagnosed sickle cell anemia. Likewise, serum proteins like glycoproteins and albumin are also analyzed using this technique. It is used in antenatal diagnosing of the beta thalassemia. (4)
2. Analysis of proteins: Different amino acids present in the proteins are separated on the basis of charge under the influence of electric current while performing cellulose acetate electrophoresis. This analysis is helpful in forensic, molecular, and clinical laboratories. Cellulose acetate membrane electrophoresis (CAME) is a classic approach to analyzing protein panels. (3)
3. Polypeptide dyes analysis: Polypeptide dyes are used in textile industries and as labeling dye in molecular biology. Using cellulose acetate electrophoresis helps analyze the nature and components of polypeptide dyes.   
4. Analysis of nucleic acid: Cellulose acetate electrophoresis is also helpful in analyzing RNA and DNA components. 

Advantages

Although a traditional method, cellulose acetate electrophoresis is not widely used. It has many advantages. Cellulose acetate electrophoresis is better than other separation methods because of its easy setup, less running time, easy detection, and precise zone formation. The advantages are described briefly below:

1. Easy setup: The instrument setup in the case of cellulose acetate electrophoresis is very simple. It does not require complicated instruments like setting gels in case of gel electrophoresis or properly maintaining capillaries for capillary electrophoresis.
2. Timely detection: There are various ways of detecting separated zones in cellulose acetate electrophoresis, like zymogram paper which is very convenient to perform. Likewise, using blotting techniques and a spectrophotometer is also very easy. 
3. Less time-consuming: The time required for completing the entire electrophoresis requires less than 2 hours. Compared to gel electrophoresis and capillary electrophoresis, the setup takes longer than 2 hours, and completion of the electrophoresis process takes about an hour. So, it consumes less time than other methods. 
4. No tailing effect: While separating the proteins, other methods can leave a tailing effect after separation. This electrophoresis separation method does not leave such effects after separation.
5. Better zone development: The zone formed after separation in cellulose acetate electrophoresis is more apparent than in paper electrophoresis. The bands are better in resolution and sharpness.

Limitations

The cellulose acetate electrophoresis is highly advantageous, but it has some limitations, which are discussed below:

1. Only applicable under the alkaline condition: The cellulose acetate electrophoresis can only be used when the buffer is alkaline. 
2. Chance of electroosmosis: The sulfonic and carboxylic acid residue can induce electroosmosis during cellulose acetate electrophoresis.
3. Difficulty in quantification: The process is difficult to quantify and requires extensive blotting techniques to quantify the amount of components after separation. 

References

1. Walker JM. Electrophoretic techniques. In: Principle and techniques of Biochemistry and Molecular Biology. New Delhi: Cambridge University Press; 2010. p. 399–432.  
2. Wild BJ, Bain BJ. Detection and quantitation of normal and variant haemoglobins: An analytical review. Annals of Clinical Biochemistry: International Journal of Laboratory Medicine. 2004;41(5):355–69.  
3. Nakayama A, Kubota R, Sakatsume M, Suzuki H, Katayama A, Kanamori K, et al. Cellulose acetate membrane electrophoresis based urinary proteomics for the identification of characteristic proteins. Journal of Clinical Laboratory Analysis. 2015;30(5):359–67.  
4. Boccacci M, Massa A, Tentori L. Application of cellulose acetate electrophoresis to globin chain separation for antenatal diagnosis of beta thalassemia. Clinica Chimica Acta. 1981;116(2):137–42. 
5. Hb A2-Pasteur-Tunis [delta59(E3)Lys–>Asn, AAG–>AAC]: a new delta chain variant detected by DNA sequencing in a Tunisian carrier of the codon 39 (C–>T) beta0-Thalassemia mutation – Scientific Figure on ResearchGate. Available from: https://www.researchgate.net/figure/Electrophoretic-study-A-Cellulose-acetate-electrophoresis-at-alkaline-pH-Lane-1_fig3_258112336 [accessed 25 Dec, 2022]