SDS PAGE Full Form
What is the full form of SDS PAGE?
Proteins can be separated based on their molecular weight using the SDS PAGE technique, which stands for Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis. The method of dividing up protein molecules according to their electrophoretic mobility is frequently used in forensics, genetics, biotechnology, and molecular biology.
Principle of SDS-PAGE
According to the SDS-PAGE principle, when a charged molecule is exposed to an electric field, it migrates to the electrode with the opposing sign. The relative mobility of charged species affects how the charged molecules are separated.
Because they encounter less resistance during electrophoresis, smaller molecules migrate more quickly. The rate of migration is also influenced by the proteins' charge and structure. The influence of the proteins' structure and the charge is eliminated by sodium dodecyl sulfate and polyacrylamide, and the proteins are separated according to the length of the polypeptide chain.
Role of SDS-PAGE
In the SDS-PAGE sample buffer, there is a detergent called SDS. Proteins' disulphide bonds are broken by SDS and some reducing agents, which causes the tertiary structure of the proteins to be disrupted.
Materials Required
Gels: These can be made in a laboratory or bought as precast gels from the market.
Electrophoresis Chambers: They help to accommodate the SDS-PAGE gels.
Protein Samples: After diluting the protein with SDS-PAGE sample buffer, the protein is boiled for 10 minutes. To prevent any tertiary protein folding, a reducing agent like dithiothreitol or 2-mercaptoethanol is also added to reduce the disulfide linkages.
SDS-PAGE Running Buffer: This is used to run the protein samples that have been loaded onto the gel.
Coomassie Stain Solution: This is used to stain and remove stains from the gel. The destaining solution is then applied to the gel, desaturating it. After that, protein bands are visible to the naked eye.
Protein Ladder: Based on the molecular size, the position of the protein of interest is found using a reference protein ladder.
Protocol of SDS-PAGE
Preparation of the GeL
For the preparation of the gel, all the reagents—aside from TEMED—are combined.
Add TEMED when the gel is ready to be poured.
The casting chamber receives the pour of the separating gel.
Before polymerization, add butanol to remove any unwelcome air bubbles.
The spaces between the glass plates are filled with the comb.
The "gel cassette" is the term for polymerized gel.
Sample Preparation
In a beaker, heat some water.
To the sample buffer, add 2-mercaptoethanol.
Add the protein sample to the buffer solution in the microcentrifuge tubes.
Place the MW markers in different tubes.
To completely denature the proteins, boil the samples for no longer than five minutes.
Electrophoresis
The electrode assembly receives the gel cassette after it has been removed from the casting stand.
The clamp stand has the electrode assembly fixed in it.
The wells of the gel are filled with 1x electrophoresis buffer, which is poured into the casting frame's opening.
In the well, pipette 30 ml of the denatured sample.
The unit is then connected to a power source and the tank is covered with a lid.
For about an hour, run the sample at 30 mA.
The bands can then be seen under UV light.
Applications of SDS-PAGE
The applications of SDS-PAGE are as follows:
It is used to calculate the molecules' molecular weights.
It is employed to determine the protein's size.
It is applied to evaluate how different structures' polypeptide compositions compare.
Used in mapping peptides.
It is employed to determine the purity of proteins.
It is utilized in protein ubiquitination and Western Blotting.
To separate the HIV proteins, it is used in HIV tests.
Used in investigating the dimensions and quantity of polypeptide subunits.
Used to examine post-translational changes.
Frequently Asked Questions (FAQs)
Proteins are separated electrophoretically through electrophoresis. Electrophoresis is a technique for separating complex protein mixtures (from cells, column fractions, subcellular fractions, or immunoprecipitation). It is also used to analyze the protein component compositions and verify the homogeneity of protein samples. It can also be used to prepare proteins for future use. During polyacrylamide gel electrophoresis, proteins move through holes in a polyacrylamide gel matrix in response to an electrical field; pore size decreases as acrylamide concentration increases. The pore size, protein shape, size, and charge all affect how quickly a protein migrates.
According to the SDS-PAGE principle, a charged molecule will migrate in the direction of the electrode that has the opposite charge when it is exposed to an electric field. The separation of charged molecules is influenced by the relative mobility of charged species. The smaller molecules migrate more quickly during electrophoresis because their resistance is lower.
Sodium dodecyl sulfate, also known as SDS, is a potent detergent that is present in high concentrations in the buffer used to prepare samples for electrophoresis. Before samples like cells can be run on a protein gel, cell membranes must be lysed and all proteins must be solubilized by SDS.
To arrange all of the protein samples that have been loaded onto the gel in a row so that they can all enter the resolving gel simultaneously, use a stacking gel. Based on their molecular weight, the proteins are separated using the resolving gel.
The most common buffer for SDS-PAGE is tris. With a pKa of 8.1, it excels as a buffer in the pH range of 7-9. It is therefore a good option for the majority of biological systems. The buffer's SDS aids in maintaining the proteins' linearity.