Cellulose Acetate Electrophoresis – Definition, Principle, Operating Procedure, Uses

Cellulose Acetate Electrophoresis is a type of zone electrophoresis technique used to separate charged biological molecules like proteins, nucleic acids and dyes in a liquid sample.

It is carried out on a porous membrane made up of cellulose acetate. This membrane is first soaked in buffer solution and then electric current is passed through it. The charged molecules move across the membrane according to their net electric charge and form sharp separate bands.

In alkaline buffer, like pH 8.6, serum proteins become negatively charged and move towards the positive electrode or anode. It is a fast method and gives clear bands with very little adsorption of proteins. The separated bands can also be made transparent and measured by densitometer.

It is mainly used in clinical laboratory for separation of serum proteins, haemoglobin variants, and some isoenzymes like LDH and CK.

Principle of Cellulose Acetate Electrophoresis

Cellulose acetate electrophoresis is based on the migration of charged molecules through a buffer soaked cellulose acetate membrane under the influence of an electric field.

The biological molecules like proteins carry net positive or negative charge depending on the pH of the buffer. When electric current is passed, the charged molecules move towards the opposite electrode. The positively charged particles move towards cathode and the negatively charged particles move towards anode.

The cellulose acetate membrane has large and uniform pores. So it shows very less molecular sieving effect. Due to this, the separation is mainly based on the net electrical charge of the molecules and not mainly on their size.

During this process, electroendosmosis also occurs. In this, positively charged buffer ions move towards the cathode and produce a backward flow of fluid. This flow opposes the normal movement of negatively charged proteins. Finally, the separated components appear as distinct bands on the membrane due to the combined effect of electrophoretic movement and electroendosmotic flow.

Materials Required for Cellulose Acetate Electrophoresis

The following are the materials required for Cellulose Acetate Electrophoresis–

1. Horizontal electrophoresis chamber – It is used to hold the buffer and cellulose acetate membrane during electrophoresis.
2. Power supply – It is used to supply constant electric current or voltage for the movement of charged molecules.
3. Cellulose acetate membrane – It is the supporting medium on which separation of proteins or other charged molecules takes place.
4. Running buffer – It is used to maintain proper pH and provide ions for conduction of electric current. Commonly barbital buffer or veronal buffer of pH 8.6 is used.
5. Filter paper wicks – These are used to connect the buffer solution with the cellulose acetate membrane.
6. Sample applicator – It is used to apply the sample on the membrane in narrow line or spot.
7. Sample well plate – It is used for keeping the samples before application on the membrane.
8. Blotting paper – It is used to remove excess buffer from the membrane surface.
9. Staining solution – It is used to stain the separated bands after electrophoresis. Example, Ponceau S or Amido Black 10B.
10. Destaining solution – It is used to remove extra stain from the membrane. Usually 5% acetic acid is used.
11. Fixative solution – It is used to fix the separated protein bands on the membrane.
12. Dehydrating agent – Absolute methanol is used for dehydration of the membrane.
13. Clearing solution – It is used to make the membrane transparent for densitometric reading.
14. Densitometer – It is used for quantitative measurement of the separated bands.
15. Control sample – Normal and abnormal control samples are used to check the accuracy of the test.

Steps / Procedure of Cellulose Acetate Electrophoresis

The following are the steps of Cellulose Acetate Electrophoresis–

1. The cellulose acetate membrane or plate is soaked in diluted running buffer for about 20 minutes. This makes the membrane saturated with buffer.
2. About 100 ml of buffer is poured into the outer compartments of the electrophoresis chamber. The filter paper wicks are placed in such a way that one end touches the buffer and other end comes over the support bridge.
3. The soaked cellulose acetate plate is removed from the buffer. It is blotted firmly with blotting paper to remove excess buffer from the surface.
4. A small amount of sample, generally 3 µL, is applied on the membrane with the help of microdispenser or sample applicator. The sample is applied as narrow band.
5. The plate is placed face down in the chamber and proper contact is made with the wicks. It is allowed to equilibrate for about 30 seconds. Then electric current is applied at about 180 volts for 15 minutes.
6. After electrophoresis, the plate is removed from the chamber. It is dipped in staining solution like Ponceau S for about 6 minutes. In this step, separated protein bands become visible.
7. The plate is washed in 5% acetic acid for removing excess stain. Usually three successive washing are done for 2 minutes each until the background becomes white and clear.
8. For densitometric study, the plate is dehydrated with methanol by two washing for 2 minutes each. Then it is treated with clearing solution for 5-10 minutes and dried in oven at 50-60°C for about 15 minutes.
9. The separated bands are observed visually. The cleared plate can also be scanned by densitometer for quantitative estimation of different fractions.

Cellulose Acetate as Electrophoretic Support Matrix

The following are the features of cellulose acetate as electrophoretic support matrix-

• Cellulose acetate is a semi-synthetic support matrix made from natural cellulose by acetylation. In this process, the hydroxyl groups of cellulose are replaced by hydrophobic acetyl groups.
• It is usually present as diacetate or triacetate form. It is made into a thin, uniform and microporous film of about 0.1 to 0.15 mm thickness.
• The pores of cellulose acetate membrane are larger than the pores of agarose gel and polyacrylamide gel. So it gives very little molecular sieving effect.
• The separation on this membrane is mainly based on the net electric charge of the molecules. It is not mainly based on the molecular size of the molecules.
• During electrophoresis, electroendosmosis also affects the movement of molecules. The buffer flow moves towards the cathode because of the negative charge present on the membrane surface.
• Cellulose acetate membrane gives rapid separation. The separation is usually completed within 15 to 60 minutes, whereas paper electrophoresis needs much longer time.
• It needs very small amount of sample. For serum protein separation, about 2 µL sample may be enough.
• It gives sharp and clear bands. The low protein adsorption and uniform pore nature reduce tailing of bands.
• After staining, the membrane can be treated with clearing solution such as methanol and glacial acetic acid. This makes the membrane transparent and the bands can be measured by densitometer.
• It is easy to handle and ready-made support medium. It does not need polymerization like polyacrylamide gel, so it is useful in routine clinical laboratory work.
• It is more costly than ordinary paper electrophoresis. But it is faster and gives better clear bands than paper method.
• It gives lower resolution than PAGE or capillary electrophoresis. It cannot separate many proteins having similar charge only on the basis of molecular weight.
• Some abnormal haemoglobins like HbS and HbD may move together on the membrane. So secondary test is needed for proper identification.
• It is commonly used for serum protein analysis. It separates serum proteins into albumin, alpha-1 globulin, alpha-2 globulin, beta globulin and gamma globulin fractions.
• It is also used for screening of haemoglobinopathies like sickle cell anaemia and thalassemia. It is also used for separation of LDH, CK isoenzymes and in some immunological tests.

Application of Cellulose Acetate Electrophoresis

The following are the uses of Cellulose Acetate Electrophoresis–

• Serum proteins are separated into five fractions. These are albumin, alpha-1 globulin, alpha-2 globulin, beta globulin and gamma globulin. The method is useful in multiple myeloma, liver cirrhosis, chronic infection and nephrotic syndrome.
• Abnormal hemoglobins are detected by this method. These include HbS, HbC and HbD. It is used in sickle cell anemia and thalassemia as a first screening method.
• Tissue specific isoenzymes are separated by this method. Lactate Dehydrogenase (LDH) and Creatine Kinase (CK) are the common examples. They are useful in detection of cardiac, skeletal muscle and hepatic injury.
• It is used in Immunoelectrophoresis (IEP) and Counterimmunoelectrophoresis (CIE). These methods are used for immunoglobulin abnormality and antigen-antibody reaction.
• In pharmaceutical field, it is used for testing of biological drugs. Protein variants, isoforms and protein contaminants are studied by this method.
• In food science, it is used for study of protein content of nutritional supplements and health products. It helps in quality testing of these products.
• In agriculture and plant pathology, protein profile is studied by this method. It is used for differentiation of strains and developmental stages of plant pathogens like Phytophthora infestans.
• Other biological fluids are also analysed by this method. These are cerebrospinal fluid, urine proteins, lipoproteins and glycoproteins.

Advantages of Cellulose Acetate Electrophoresis

The following are the advantages of Cellulose Acetate Electrophoresis–

• It is rapid method. Separation is completed within 15-60 minutes. In filter paper method, the separation needs about 12-14 hours.
• The bands are sharp and clear. The membrane has uniform pore structure. Protein absorption is also very low, so tailing effect is almost absent.
• The membrane can be made transparent by clearing solution. So the separated bands can be measured by optical densitometer.
• Very small amount of sample is needed. About 2 µL serum is enough for analysis. So it is useful when sample amount is less.
• The membrane is ready made. It only needs soaking in buffer before use. So the method is simple and suitable for many samples.
• After staining, clearing and drying, the membrane forms a tough plastic film. It can be stored for long time as a permanent record.
• It is simple and economical method. It does not need very complex apparatus like capillary electrophoresis or polyacrylamide gel electrophoresis.
• It is safe and easy to perform in clinical laboratory. The technique is not very difficult and gives reproducible result.
• It has broad use in clinical and biological samples. It is used for separation of serum proteins, hemoglobins, isoenzymes, glycoproteins and also in immunological techniques.

Limitation of Cellulose Acetate Electrophoresis

The following are the limitations of Cellulose Acetate Electrophoresis–

• Resolution is lower in this method. The membrane pores are large and less uniform. So molecular sieving effect is very less than polyacrylamide gel electrophoresis and capillary electrophoresis.
• Closely migrating variants are not separated clearly. Some hemoglobin variants like HbS and HbD, or HbC and HbE may move almost same position. So other test like HPLC is needed.
• Induced electroosmosis may occur in the membrane. This is due to presence of trace sulfonic and carboxylic residues. It causes back flow of buffer and opposes the normal movement of proteins.
• Mechanical stability of the membrane is low. So handling must be done carefully. Reproducibility is also less than polyacrylamide gel.
• It is sensitive to heat. During electrophoresis Joule heating is produced. If temperature is not controlled below about 23°C, buffer may evaporate and protein precipitation or artifacts may occur.
• Automation is limited in this method. Most of the steps are manual or semi-automatic. So throughput is less than fully automated capillary electrophoresis.
• Cost of membrane and procedure may be relatively high. So it may not be suitable for all routine laboratories.
• Application is limited for some works. Proteins are separated mainly by charge, not by molecular weight. So it is mostly useful in routine clinical serum analysis, hemoglobin study and some specific electrophoretic tests.

Comparisons with Gel or Capillary Electrophoresis

The following are the comparisons of Cellulose Acetate Electrophoresis with other electrophoretic methods-

1. Cellulose Acetate Electrophoresis is faster than filter paper electrophoresis. It takes less than one hour. But in filter paper electrophoresis, about 12-14 hours is needed.
2. In Cellulose Acetate Electrophoresis, bands are more sharp and clear than filter paper method. Protein adsorption is very low. So tailing of protein is almost absent.
3. Cellulose Acetate Electrophoresis membrane can be made transparent by clearing solution. So bands are measured by densitometer. But filter paper remains opaque and measurement is not so easy.
4. In this method, sample volume is very low. About 2 µL sample is needed. But filter paper electrophoresis needs more sample, about 20-50 µL.
5. Polyacrylamide Gel Electrophoresis (PAGE) gives higher resolution than Cellulose Acetate Electrophoresis. In PAGE, pore size can be controlled and proteins are separated by molecular weight. But in Cellulose Acetate Electrophoresis, separation is mainly by electrical charge.
6. Cellulose Acetate Electrophoresis is easier and safer than PAGE. It uses ready-made membrane. It does not need preparation of acrylamide gel, which is toxic in monomer form.
7. PAGE is more useful for molecular weight determination and protein sequencing work. But Cellulose Acetate Electrophoresis is more useful for routine clinical protein separation because it is faster and simple.
8. Capillary Electrophoresis (CE) is more rapid and fully automated than Cellulose Acetate Electrophoresis. It can complete analysis within very short time, sometimes below 10 minutes.
9. Capillary Electrophoresis needs costly instrument and trained person. But Cellulose Acetate Electrophoresis is simple, low cost and useful in low resource laboratory.
10. Cellulose Acetate Electrophoresis gives permanent physical record after staining, clearing and drying of membrane. But Capillary Electrophoresis gives only digital trace.
11. Agarose Gel Electrophoresis (AGE) gives good resolution and can be used with moderate automation. Cellulose Acetate Electrophoresis also gives good separation for routine work, but it is more simple in handling.
12. Agarose Gel Electrophoresis is commonly used for many nucleic acid and protein separation work. But Cellulose Acetate Electrophoresis is more common in routine clinical separation of serum proteins, hemoglobins and some other charged molecules.

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