SDS Page

SDS PAGE ,also known as Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis is a technique used for separating the proteins based on their molecular weight. It is a widely used technique in forensics, genetics, biotechnology, and molecular biology to separate the protein molecules based on their electrophoretic mobility.

Principle of SDS-PAGE

The principle of SDS-PAGE states that a charged molecule migrates to the electrode with the opposite sign when placed in an electric field. The separation will take place as the mobility of the charged species. The tiny molecules tend to move faster due to their less resistance at the time of electrophoresis. The rate of migration influences the structure and the charge of the protein. 

Sodium dodecyl sulphate and polyacrylamide help to eradicate the influence of structure and charge of the proteins, and the proteins are separated based on the length of the polypeptide chain.

Function of SDS in SDS-PAGE

SDS can be defined as a detergent present in the SDS-PAGE sample buffer. The function of SDS is to break the disulphide bonds of proteins disrupting the tertiary structure of proteins along with some reducing agents.

Materials Required

• Power Supplies: It is used to convert the AC current to DC current.

• Gels: These are either self prepared in the laboratory or are purchased from the market.

• Electrophoresis Chambers: The chambers of the SDS-PAGE gels that fit well should be used.

• Protein Samples: The protein is dissolved with an SDS-PAGE sample buffer and boiled for 10 minutes. A reducing agent such as dithiothreitol or 2-mercaptoethanol is also added to minimize the disulfide linkages to prevent any tertiary protein folding.

• Running Buffer: The protein samples filled on the gel are run in SDS-PAGE running buffer.

• Staining and Destaining Buffer: Coomassie Stain Solution is used to stain. A destaining solution is used to destain a gel. Protein bands can then be observed with naked eyes.

• Protein Ladder: A reference protein ladder is used to locate the protein of interest based on the molecular size.

Protocol of SDS-PAGE

Preparation of the Gel

• All the reagents are combined, except TEMED, for the preparation of gel.

• When the gel is ready to be put, add TEMED.

• The gel used to separate is poured in the casting chamber.

• You need to put butanol before polymerization to remove the unwanted air

• bubbles present.

• Between the glass plate, the comb is inserted. 

• The “gel cassette” is the  polymerized gel. 

• Sample Preparation

• Boil some water in a beaker.

• Add 2-mercaptoethanol to the sample buffer.

• Put the buffer solution in microcentrifuge tubes and add protein samples to it.

• Take MW markers in separate tubes.

• Bring the samples to boil for less than 5 minutes to completely denature the proteins.

Electrophoresis

• The gel cassette is put off from the casting stand and placed separately in the electrode assembly.

• The clamp stand is fixed to the electrode assembly.

• a 1X electrophoresis buffer is added to the opening of the casting frame to fill the wells of the gel.

• Fit 30ml of the denatured sample in the well.

• The tank is covered with a lid and the unit is attached to a power supply.

• The sample is made to run at 30mA for about 1 hour.

• UV light is used to observe the light.

What is meant by SDS PAGE?

It is an electrophoretic system that was developed by Laemmli. The system is a system that is primarily used as a method to separate proteins. With the help of this electrophoretic system, we can separate proteins that have molecular masses of 5 to 250 kDa. The system is also used to attain the separation of proteins that are of complex mixtures. It will affect the molecular structure of the proteins that will undergo the process of electrolysis. The electrophoretic system is used to attain high-resolution separation of proteins.

Why is SDS PAGE used?

The system includes the method of passing current and making the proteins offer sample travel through the gel towards the electrode. This entire process is known as SDS Page. It separates the proteins depending on the mass. The ionic detergent that is found in the SDS denatures and gets attached to the protein masses and makes them uniformly negative.

There is a significant difference between the page and SDS page because of the difference between the factor on which the protein migration rate depends. In the case of a page, the separation of the protein depends on the mass and structure of the sample. But, in the case of the SDS page, the protein migration depends only on the mass of the protein molecules.

The Advantages of SDS PAGE

The electrophoretic system, namely the SDS page, helps us to perform a lot of complicated tasks as it simplifies the process of operation. Performing the system properly can help us to determine the molecular weight of protein in a sample. The process also helps us to detect specific proteins on the basis of the characters and mass. Most importantly it helps us to identify different species on the basis of the mass of the protein.

Other systems also performed, known as gel electrophoresis. It helps us to separate the macromolecules of a particular sample by producing an electric field. But the SDS page is a form of gel electrophoresis system that is of high resolution. It is an advanced system that is used to separate protein molecules solely based on mass.

Limitations 

There are some limitations associated with the concept and applying the methods of the SDS page when it comes to identifying a particular strain on the basis of its DNA or RNA. Since the electrophoretic system helps us to visualise the protein molecules and the structures as it can identify the mass of the proteins, it is not helpful to identify the DNA or RNA structure of a strain. DNA and RNA are primarily composed of nucleic acids and when a component is contaminated with nucleic acid, the structure will not be visible on the SDS page gel.

In the system of SDS page, the SDS refers to an anionic detergent that is primarily used to denature or take away the basic characteristics of protein molecules in a sample. The SDS, which is negatively charged, will destroy the structures of protein molecules and then it will be strongly attracted toward the node of the electric field. This is how it helps us to separate the proteins depending on the mass.

The SDS page cannot determine the purity of the protein molecules. To test the purity, we have to perform various quantification methodologies like UV-Vis. Systems like electrophoresis are generally used to visualise the protein structure and separate the protein molecules on the basis of the mass or shape and structure. That is why pointing out the amount of impurity present in a protein molecule is not possible with the help of performing SDS-page or any other electrophoretic system.

Components Present in SDS Page

SDS is known as sodium lauryl sulfate sodium dodecyl sulfate. The system is primarily made with one of these previously mentioned components and gel that is known as a polyacrylamide. A particular temperature range and a specific amount of heat play an important role in carrying out the entire process of the separation of protein molecules. The gel is of prime importance as it takes away basic characteristics of the protein molecules so that the mass of the protein molecules do not get influenced by the structure and charge. Then the protein molecules are separated depending on the polypeptide chain length.