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The SDS-PAGE is a very important topic that gives a way to separate the proteins according to their masses. The system was developed by Ulrich K. Laemmli, a professor at the University of Geneva.
The technique is used in forensics, genetics, and biotechnology. The SDS can also be defined as a detergent that is present in the SDS-PAGE sample buffer.
What is SDS PAGE?
SDS PAGE is an electrophoretic system (Discontinuous) that was developed by Ulrich K. Laemmli. This is very commonly used to separate molecular-mass proteins between 5 and 250kDa. The combination of SDS and Polyacrylamide gel helps eliminate the influence of the structure and the charge and proteins get separated solely according to their molecular weights. SDS PAGE full form is Sodium dodecyl-sulfate-polyacrylamide gel electrophoresis.
The complete system is made of sodium lauryl sulfate sodium dodecyl sulfate and the gel, polyacrylamide. The entire process needs to be done at a particular heat. The gel has prime importance as it takes away the basic characteristics of the protein molecules. The protein molecules get separated based on their polypeptide chain length.
What are the applications of SDS PAGE?
There are many applications of SDS-PAGE:
It is used in peptide mapping
It analyzes the post-translational modifications.
It is used in separating the HIV proteins in the HIV tests.
It is used in measuring the size of the protein
It is used in the eradication of the pureness of proteins.
Limitations of SDS-PAGE
Some limitations are related to this concept. The Electrophoretic system helps us to visualize the protein and its structure, but it is not helpful at all when used in the identification of RNA or the DNA structure of the strain. As we know, DNA and RNA are both the composition of Nucleic acids. So, when the component is contaminated with nucleic acids the structure will not be visible on the SDS PAGE gel.
The SDS is considered an anionic detergent that is used in the denaturation of the basic characteristics of any protein molecule. It is negatively charged and destroys the structure of the molecule when it is strongly attracted toward the node of the electric field.
Materials used in the SDS-PAGE
Given below are the materials used in the SDS-PAGE:
Power Supplies: converts the AC to DC
Gels: Prepare by self or bought from the market.
Protein Samples: The protein is dissolved with an SDS-PAGE gel and boiled for about 10 minutes. A reducing agent is also used to minimize the disulfide linkages. (Dithiothreitol or 2-mercaptoethanol)
Electrophoresis Chambers: Best fitted chambers should be used.
Running the Buffer: The sample of the proteins is filled on the gel.
Protein Ladder: It is used in locating the protein of interest according to their molecular size.
Staining and Destaining Buffer: Coomassie Stain Solution is used in staining, also the protein bands can be observed with the naked eyes.
SDS PAGE Electrophoresis
SDS PAGE electrophoresis method allows the separation of proteins according to their masses. The discontinuous gel used is polyacrylamide-based, this gel is sandwiched between the two glass plates.
The amount of SDS used is 1.4 grams to a gram of protein. SDS mainly acts as a surfactant as it helps in conferring the proteins with a charge-to-mass ratio. The voltage used is 100 V, 10-20 V per cm gel length this whole process lasts for about half an hour to several hours. It depends upon the voltage and the length of the gel that is being used. By the optical control of the migrating-colored band, electrophoresis can be stopped, before the dye and also the whole sample has been completely migrated through the gel
SDS PAGE Principle
SDS PAGE principle says that the charged molecule migrates to the electrode side when it is in an electric field. The structure and the charge of the protein get influenced by the rate of migration. The Sodium dodecyl sulfate and polyacrylamide help in eradicating the influenced structure and the charge of the proteins.
The separation takes place according to the mobility of the charged species; the tiniest molecules seem to move faster due to their lesser resistance during the time of electrophoresis.
Difference between Native PAGE and the SDS PAGE
Native PAGE: This uses non-denatured gels in the separation of the proteins. Here, the conformation, folding, and amino acid chains of the proteins are the main factors upon which the whole process of separations depends. The proteins here get separated based on their charge, size, and shape. The proteins are stable here and also be recovered later.
SDS PAGE: This technique uses denatured gel and separates the proteins based on their masses. SDS is added to the gel which imparts a charge on the samples of the protein. The proteins are not at all stable in SDS-PAGE which is why they cannot be recovered.
Conclusion
SDS-PAGE is (Sodium dodecyl sulfate Polyacrylamide gel Electrophoresis) as mentioned above is a discontinuous electrophoretic system that helps in the separation of the proteins with the help of their molecular masses. The given FAQ section will help in getting the answers to the most probable queries that might arise and also will provide additional information which will aid a better understanding of the topic.
