SDS Page – Its principle, function, and application

History:

     The discovery of the electrophoresis principle, which describes the movement of charged and dissolved atoms or molecules in an electric field, earned Arne Tiselius the 1948 Nobel Prize in Chemistry. The separation was improved by using a solid matrix (originally paper discs) in a zone electrophoresis. The separation via the stacking effect could be improved thanks to L. Ornstein and B. J. Davis’ discontinuous electrophoresis from 1964.

 In comparison to the previously employed paper discs or starch gels, the use of cross-linked polyacrylamide hydrogels gave a higher stability of the gel and prevented microbial degradation. In 1965, David F. Summers, a member of James E. Darnell’s working group to separate the proteins of the poliovirus, first described the denaturing impact of SDS in continuous polyacrylamide gels and the ensuing improvement in resolution. To characterize the proteins in the T4 bacteriophage’s head, Ulrich K. Laemmli first described the modern SDS-PAGE technique in 1970.

 Many people credit this Laemmli paper with developing contemporary SDS-PAGE, although Jake Maizel came up with the idea while on sabbatical in the MRC lab at the time Laemmli started working there as a postdoctoral fellow. With Laemmli’s help, Maizel and his previous technology underwent additional development. Laemmli and Maizel had intended to publish a Methods paper as a follow-up, but this never happened. Maizel provides a quick perspective on the evolution of SDS-PAGE.

Introduction of SDS page:

Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis, or SDS PAGE, is a method for sorting proteins according to their molecular weight. In forensics, genetics, biotechnology, and molecular biology, it is a common practice to separate protein molecules according to their electrophoretic mobility.

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 charged species’ movement will cause the separation to occur. Due to their lower resistance during electrophoresis, the small molecules tend to move more quickly. The structure and charge of the protein are affected by the rate of migration.

Protein structure and charge are eliminated with the aid of sodium dodecyl sulfate and polyacrylamide, and the proteins are sorted based on the length of the polypeptide chain.

The function of SDS in SDS-PAGE:

   A detergent that is present in the SDS-PAGE sample buffer is known as SDS. Along with some reducing chemicals, SDS can alter the tertiary structure of proteins by rupturing their disulfide linkages.

Material Required:

Power supplies:

 They are employed to transform AC electricity into DC.

Gels:

These can be made in a laboratory on your own or bought off the shelf.

Electrophoresis Chambers:

 SDS-PAGE gels that fit well in the electrophoresis chambers should be utilized.

Protein samples:

The protein is boiled for ten minutes after being dissolved in an SDS-PAGE sample buffer. To reduce the disulfide bonds and avoid any tertiary protein folding, a reducing agent like dithiothreitol or 2-mercaptoethanol is also included.

Running Buffer:

SDS-PAGE running buffer is applied to the protein samples that have been loaded onto the gel.

Staining and Destaining Buffer:

  Using Coomassie Stain Solution as a staining and destaining buffer. To remove stains from a gel, apply a destaining solution. Then, it is possible to see protein bands with the naked eye.

Protein Ladder:

 Based on the molecular size, the protein of interest is located using a reference protein ladder.

Explain the protocol of SDS PAGE.

Sample preparation:

1. Wear gloves at all times.
2. Make a 100°C hot water bath. Put some water in a 600 mL or larger beaker and heat it in the microwave or on a hot plate. (This could take up to 15 minutes.)
3. Combine 20 mL of Laemmli sample buffer/Loading with 10 mL of each protein sample. dye in screw-top microcentrifuge tubes with labels.
4. To completely denature the proteins, boil the samples for no longer than 5 minutes.
5. The sample tubes should be kept at room temperature after boiling until they are ready to be loaded onto the gel.

Preparation of the gel and electrophoresis chamber:

1. Wear gloves at all times.
2. The pre-cast gel should be taken out of its box. Remove the green strip off the gel’s bottom with care.
3. Open the vertical gel cassette assembly’s two green side clamps.
4. On one side of the cassette, use the pre-cast gel, and on the other, use the clear buffer dam. Next, carefully close the clamps on the green side.
5. The color of the electrodes (red and black) should match the colour guides at the sides of the vertical gel chamber as you insert the cassette.
6. The wells should be completely submerged in 1X Tris Glycine SDS PAGE buffer inside the cassette.
7. 1X Tris-Glycine SDS PAGE buffer should be added to the bottom of the vertical gel chamber until it reaches the mark on the side for one to two gels.

Loading the gel into the gel:

• On the cassette’s top, place the yellow gel loading guide.
• 10 L of the produced protein MW standard should be pipetted into the first (#1), fifth (#5), and final (#10) lanes using gel loading tips.
• Add 10 L of each protein sample to the remaining wells (2-4; 6-9) of the gel using micropipettes equipped with gel loading tips. In your notebook, make a note of the samples that are in each lane.

Electrophoresis:

1. Cover the vertical gel chamber with its lid.
2. Connect the red and black wires to the power supply’s appropriate matching coloured terminals.
3. Connect the power supply, then flip on the switch.
4. After choosing “Constant Voltage,” increase the voltage to 300 volts.
5. Hit the “run” key.
6. Set a 10-minute timer.
7. Turn off the power and halt the run if the protein marker’s smallest band has descended to 1 cm from the gel’s bottom edge; if not, keep running until it does.
8. Remove the gel chamber’s power supply and connections.
9. The gel chamber should be properly disassembled and removed.
10. Do not pour used buffer down the sink; instead, place it in a used buffer container.
11. The gel must either go via staining or being imaged by using a gel documentation camera system.

Why is SDS PAGE used?

The device employs a technique for conducting current that causes a sample of proteins to move through the gel and toward an electrode. The complete procedure is referred to as SDS Page. According to the mass, the proteins are separated. The SDS’s ionic detergent denatures the protein masses, attaches to them, and renders them uniformly negative.

Because the factor that determines the protein migration rate is different between the page and the SDS page, there is considerable variation between the two. The mass and structure of the sample play a role in the separation of the protein in the case of a page. However, in the case of the SDS page, the movement of the proteins is solely based on their mass.

What are the applications of SDS PAGE?

1. It is employed to calculate the molecules’ molecular weights.
2. It is employed to determine the protein’s size.
3. When mapping peptides
4. It is applied to evaluate how different architectures’ polypeptide compositions compare.
5. It is employed to determine the proteins’ purities.
6. It is utilized in protein ubiquitination and Western Blotting.
7. To isolate the HIV proteins, it is utilized in HIV tests.
8. Investigating the dimensions and quantity of polypeptide subunits.
9. To examine post-translational changes.

What are the advantages of SDS PAGE?

The electrophoretic system, namely the SDS page, facilitates the completion of numerous challenging jobs by streamlining the operational process. The system’s appropriate operation can assist us in determining the protein’s molecular weight in a sample. The method aids in the identification of particular proteins based on their characteristics and mass. The most essential benefit is that it enables us to distinguish between various species based on the mass of the protein.

Additionally, technologies called gel electrophoresis were used. By creating an electric field, it aids in the separation of the macromolecules in a given sample. However, the SDS page uses a high-resolution kind of gel electrophoresis. It is a sophisticated system that divides protein molecules exclusively based on mass.

References:

https://www.vedantu.com/biology/sds-page

https://byjus.com/biology/sds-page/

https://en.wikipedia.org/wiki/SDS-PAGE

https://bio.libretexts.org/Bookshelves/Biotechnology/Lab_Manual