ELECTROPHORESIS

INTRODUCTION

Electrophoresis is a technique based on the mobility of ions in an electric field. Positively charged ions migrate towards a negative electrode (cathode) and negatively charged ions migrate toward a positive electrode (anode). For safety reasons one electrode is usually at ground and the other is biased positively or negatively. Ions have different migration rates and can therefore be separated. Mobility of a particle is directly proportional to the voltage applied and the net charge of a particle, while it is inversely proportional to the friction offered by the particle in electric field depending upon molecular size and shape.

INSTRUMENTATION

The apparatus consists of a high-voltage supply, electrodes, buffer, and a support for the buffer such as filter paper, cellulose acetate strips, polyacrylamide gel, or a capillary tube. Open capillary tubes are used for many types of samples and the other supports are usually used for biological samples such as protein mixtures or DNA fragments. After a separation is completed the support is stained to visualize the separated components. Resolution can be greatly improved using isoelectric focusing. In this technique the support gel maintains a pH gradient. As a protein migrates down the gel, it reaches a pH that is equal to its isoelectric point. At this pH the protein is neutral and no longer migrates, i.e., it is focused into a sharp band on the gel at that point.

MEDIA FOR ELECTROPHORESIS

1. Paper (obsolete)
2. Cellulose acetate membrane (CAM).
3. Gels

1. 1. 1. Starch Gel
      2. Polyacrylamide Gel (PAGE)
      3. Agar Gel
      4. Agarose Gel

Cellulose Acetate Membrane (CAM) Electrophoresis

INTRODUCTION:

Cellulose Acetate Membrane (CAM) electrophoresis is a widely used laboratory technique for separating and analyzing proteins, haemoglobins, lipoproteins, and isoenzymes. It helps in identifying abnormal patterns of these molecules in various disease processes and enables the quantitation of normal and abnormal bands. This versatile technique provides valuable insights into various biochemical and clinical applications.

APPARATUS:

• An electrophoresis chamber with two compartments separated by a partition.
• Inert electrodes (e.g., platinum) in each compartment.
• Equal amounts of suitable buffer solution in both compartments.
• A bridge across the partition to hold a membrane or gel.
• Sample application area on the membrane.
• Electrical power source with polarity indicator.
• Prescribed voltage and current settings for electrophoresis.

REAGENTS:

• Cellulose acetate strips of appropriate size.
• Barbitone buffer (pH 8.6, ionic strength 0.05): Dissolve 10.16g sodium barbitone and 1.84g diethylbarbituric acid in 800 ml water, make up to 1L.
• Fixative solution: Dissolve 5g trichloroacetic acid (TCA), 5g zinc sulfate (ZnSO4), and 0.35g sulphosalicylic acid (SSA) per 100 ml distilled water.
• Ponceau S (0.5% w/v in 5% trichloroacetic acid). Comassie Brilliant Blue (CBB) or Amido Black can also be used as protein stains.
• Acetic acid (5% v/v in water) for destaining.
• Clearing solution: Mix 15 ml glacial acetic acid with 85 ml methanol (prepare with caution due to its corrosive nature).

PROCEDURE:

1. Mark cellulose acetate strips and soak them in the running buffer to remove air bubbles.
2. Lightly blot strips to remove excess buffer.
3. Place strips over the bridge in the electrophoresis tank and insert filter paper wicks at both ends, dipping into the buffer.
4. Apply 3-5 µl of the sample near the cathode in a row, leaving spaces between spots and margins.
5. Replace the lid and connect the power supply. Adjust the current to 0.4 mA per cm width of the strip (~185V) and run for 20-60 minutes.
6. After electrophoresis, remove the strip, trim it, and soak it for 5-10 minutes in the fixative solution (10% TCA).
7. Submerge the strip in Ponceau S solution for 10 minutes for staining.
8. Destain the strip using several changes of acetic acid.
9. For densitometry, the strip can be used as-is or cleared with a dip in the clearing solution and dried at 60-80°C.

USES:

• This method significantly improves our capacity to diagnose diseases by identifying irregular patterns of plasma proteins in various disease processes.
• Quantification of normal and abnormal protein bands.
• Identification and quantification of normal and abnormal haemoglobins.
• Quantitation of lipoproteins.
• Identification of isoenzymes.

Cellulose Acetate Membrane Electrophoresis is a valuable tool in clinical and biochemical research, aiding in the diagnosis and understanding of various medical conditions and protein-related disorders.

SDS-PAGE

It stands for sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE) and is useful for molecular weight analysis of proteins. SDS is a detergent that dissociates and unfolds oligomeric proteins into its subunits. The SDS binds to the polypeptides to form complexes with fairly constant charge to mass ratios. The electrophoretic migration rate through a gel is, therefore, determined only by the size of the complexes. Molecular weights are determined by simultaneously running marker proteins of known molecular weight.