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Definition
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Amino acids, nucleotides, polypeptides, and other compounds in a colloidal state can be separated by the application of external voltages which cause charged colloid particles to move toward an electrode. This phenomenon is known as electrophoresis, from the Greek, "borne by electricity." This phenomenon of movement of electrically charged particles in a fluid under the influence of an electric field also called Cataphoresis, the. If the liquid rather than the particles is set in motion-e.g., through a fixed diaphragm-the phenomenon is called electroosmosis.
Electrophoresis is used to analyze and separate colloids (e.g., proteins) or to deposit coatings, as on elements used in electron tubes. Electrophoresis is a commonly used technique to separate proteins, lipoproteins, nucleic acid, particles, emulsion grains, or even bacteria on the basis of their net charge in specified buffered media. The electrophoretic phenomenon is especially important in food science for the study and fractionation of proteins. For many years, electrophoresis technique is responsible for the discovery and separation many major proteins such as enzymes, hormones, and antibodies.
In addition, electrophoresis is easy to perform and provides a high resolution. Its cost is relatively low and is commonly used at the preparative level. Furthermore, it furnishes information on the charge, conformation, and the shapes of the analytes.
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Principle of electrophoresis
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Movement of proteins depends on various aspects:
Charges on the proteins:
Proteins are sequence of amino acids that can be ionized depend on their acid or basic character. The N- and C- terminal and T-groups of the polypeptide can be ionized, contributing to the overall charge. The protein's net electric charge is the sum of the electric charges found on the surface of the molecule as a function of the environment.
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At the pI of a specific protein, the protein molecule carries no net charge and does not migrate in an electric field.
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At pH above the pI, the protein has a net negative charge and migrates towards the anode.
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At pH below the pH, the protein obtains a net positive charge on its surface and migrates towards the cathode.
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Depending on the pH of the buffer, proteins in a sample will carry different charges. When an electric field is applied, proteins will migrate towards their corresponding poles. The rate of migration will depend on the strength of their net surface charges:
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rate of migration will depend on the strength of their net surface charges: |
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The protein that carries more +ve charges will move towards the cathode at a faster rate.
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The protein that carries more -ve charges will move towards the anode at a faster rate.
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In this regard, proteins can be separated based on their electric charges. |
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Shape of the Proteins:
The two proteins below carry the same charges. Still they be separated by electrophoresis.
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When starch or polyacrylamide gels are used, proteins can also be separated based on their difference in shape. A long, loose protein tends to interact more with the gel network and travels at a slower rate than a globular protein.
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Size of the Proteins:
SDS Polyacrylamide Gel Electrophoresis
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The proteins below have the same charge but are different in size and yet they can they be separated.
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The use of SDS-PAGE enables the separation of proteins by size.
Docium dodecyl sulfate (SDS) is used as a detergent in electrophoresis to dissociate a protein.
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SDS are attached to the protein in a constant ratio. Proteins now have identical charge density.
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SDS-polypeptide complex assumes a rod-like shape. |
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The proteins are now ready to be separated based on the difference in shape.
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The larger protein will interact with the gel structure; thus, travels slower in the gel. It is therefore possible to get an estimation of molecular mass by comparing the migration distance to that of a protein with a known mass.
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log (MASS) = k (Migration Distance)
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Electric Field
The rate of separation (v) quickly depends on the electrophoretic mobility (U) and electric field strength (E):
v = U * E
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How can we improve the efficiency of electrophoresis?
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increase U and E
We can increase the E by increasing the electric current. However, it will also introduce heat.
An efficient cooling system is therefore essential in an electrophoresis system.
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