SDS-PAGE: Introduction, Principle, Working and Steps

Introduction

A technique used for the separation of proteins based on their molecular weights is known as (SDS-PAGE) sodium dodecyl sulfate-polyacrylamide Gel electrophoresis.

Principle of SDS-PAGE

The principle states that the molecules separated based on their electrophoretic mobility will migrate towards their respective electrodes when placed in an electric field.

Electrophoretic mobility depends on the molecule’s structure, shape, and charge.  The structure and charge of the proteins are mainly responsible for the migration. The smaller molecules will move faster due to less hindrance whereas larger ones will move slower due to greater hindrance. Thus, molecules are only separated based on their molecular weights.

Material required

• Power supplies: The power supply is used to convert AC into DC.
• Gels: The gels used are either prepared in the laboratory or bought from the market in which the protein travels.
• Electrophoresis chambers: The chambers in which SDA-PAGE gels pours and protein are separated.
• Protein sample: The Protein sample used is first diluted using SDS-PAGE sample buffer, boiled for 10 minutes. Then a reducing agent is used to prevent the formation of disulfide bonds forming a tertiary structure.
  
• Running buffer: Buffer inside the chamber carries ions and current that maintains specific pH.
  
• Staining and de-staining Buffer: Stains are used to visualize the proteins
• Protein ladder: A protein ladder is used as a standard to estimate the size of the protein separated in the electrophoresis.

Agents used in SDS-PAGE

β Mercaptothion / dithiothreitol (DTT)

The disulfide bridges present between the polypeptide chains are responsible for the secondary structure of the protein. However, β Mercaptoethanol breaks the disulfide bonds that are present in the protein structure. When bonds are broken down then the S-S bonds become -SH, -SH bonds leaving the peptide end free. Therefore, on treating with β Mercaptoethanol the protein will have only a primary structure and be separated based on its size in the gel.

Sodium dodecyl sulfate (SDS)

SDS is used in the preparation of sample buffer to impart a negative charge on the protein. After the treatment with β Mercaptoethanol, all the proteins are in a linear structure so that SDS will impart a negative charge and all the proteins will move towards the positive electrode. Thus, separated only on their molecular size. Moreover, SDS is also present in the gel making sure that all the proteins should stay negative throughout the gel.

Bromophenol Blue (BPH)

Bromophenol blue is used as a tracking dye in the electrophoresis as it imparts color in the protein being separated in the gel. It is also mixed with a sample solution. It has a slightly negative charge so that it could reach the positive electrode before the protein molecule. It indicates the migration of protein towards the electrode.

Polyacrylamide gel

When the monomer acrylamide in water polymerizes with a small amount of a cross-linker e.g., N, N’-Methylenebisacrylamide, the polyacrylamide gel is manufactured. Both co-polymerize and makes the 3D network of straight-chain in which acrylamide with diacylamine are interconnected.

However, the other cross linkers that are used instead of bisacrylamide are:

• Piperazine diacrylate (PDA): It is used to reduce silver stains in the background in SDS-PAGE.
• N, N’-diallyltartardiamide (DATD): It is a disruptable cross-linker that solubilizes the gel.

However, the ratio of acrylamide and bisacrylamide determines the size of pores in the gel. So, in a discontinuous gel system, different concentration of pH is used for resolving and stacking gel.

Working of stalking gel

The proteins are separated on the interface of stacking and resolving gel. So, the stacking gel stacks the protein in the gel and runs separating gel approximately at the same time no matter what is the size of the protein is.

Two types of molecules are responsible for this purpose:

• Trailing Glycine molecules from tris-Glee electrophoresis buffer.
• Leading chloride ions from Tris-HCL running buffer.

When the voltage is applied, the migration of molecules occurs. Glycine is the simplest amino acid residue present in the form of zwitter-ion in stacking gel and their electrophoretic mobility is very low as compared to chloride ions. So, chloride ions will move faster as compared to the glycine residues.

In this way, a steep voltage gradient is formed between chloride and glycine ions. Thus, proteins reside somewhere between the chloride ions (faster one) and glycine residue (slower one) and the sample becomes “stalked” into a very thin, distinct layer in order of electrophoretic mobility.

The interface between stacking gel and resolving gel

When molecules reach the interface between stacking and resolving gel the pH increases and the pore size decreases abruptly. When increases the glycine residue will no longer be in the form of zwitter-ion. They become ionized and move faster in the stacking gel. Chloride ions will travel in no time and leaving the protein molecules to be separated based on their molecular sizes.

Summary of Gels

	Stacking gel	Resolving Gel
Polyacrylamide concentration	Low	High
Pore size	larger	Smaller
The pH of Tris-Cl- used	6.8	8.8
Purpose	To stack the polypeptides on the interface of stacking gel and resolving gel.	To separate the polypeptides solely based on size.
Electrophoretic mobility	Glycine<protein mixture< BPB<CL–	Protein mixture<glycine<BPB<CL–

Ammonium persulfate and TEMED

Polymerization of the gel occurs by a free radical mechanism. TEMED (N, N, N, N-tetramethyl ethylenediamine) acts as a catalyst and generates free radicals of sulfate. Then these sulfates for free radical generation are provided by ammonium persulfate

Major steps of SDS-PAGE

Poring of the resolving gel:

Resolving gel is poured between two glass plates (called one short, one tall) clipped together on a casting frame. Isopropanol is added to the top of the gel to remove bubbles. (The level of the gel is predetermined by placing the comb on the glass plates and leaving approximates of 1cm space below the comb. The pen is used to mark the level, and gel is poured up to this mark). The gel is allowed to solidify. When the gel is solidified, isopropanol is removed by filter paper.

Pouring of the stacking gel:

The solidification of resolving gel, loading of stacking takes place. The comb is placed immediately after loading. The gel is allowed to polymerize. After solidification of stacking gel, the comb is removed carefully so as not to damage the well.

Loading the ladder in the wells:

The ladder is added into the well very carefully by using a micropipette. The ladder is mostly pre-stained with the known molecular weight protein.

Loading of samples in the well:

Samples are loaded in each sample with an equal amount of the protein’s mixture using a micropipette. Loading of the sample should be careful, not to damage the well or sample may come out the well due to extra filling. At this stage, the sample will become blue due to (bromophenol).

Gel runs by applying the voltage:

Voltage is applied after dipping the sandwich gel and glass plate in the running buffer. Voltage should be removed when the tracking dye reaches the gel.

Subsequent analysis – (Coomassie blue staining):

The gel is rinsed with deionized water 3-5 times to remove SDS and buffer. It may create a hindrance with the Coomassie blue stain to the proteins. Then the gel in the blue stain is shaken in an incubator a room temperature. The invisible bands of proteins begin to appear within minutes and took almost 1 hour to complete.

Reference and Sources

• https://www.vedantu.com/biology/sds-page
• https://www.gurukpo.com/Content/model-paper-2019/science/M.Sc.%20Previous%20Year%20(BT)-%20Biological%20Macromolecules,%20Enzymology%20and%20Techniques.pdf
• https://collegedunia.com/exams/sds-page-principle-applications-limitations-and-gel-electrophoresis-biologyarticleid-3733
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3456489/
• https://bitesizebio.com/580/how-sds-page-works/
• https://www.coursehero.com/file/16321207/SDS-Page/
• https://www.researchgate.net/publication/224829868_Gel-Electrophoresis_and_Its_Applications
• https://www.researchgate.net/publication/269713118_Methyleneation_of_Peptides_by_N_N_N_N_-Tetramethylethylenediamine_TEMED_under_Conditions_Used_for_Free_Radical_Polymerization_A_Mechanistic_Study
• https://www.coursehero.com/file/107363059/MACUCWAKC-30306124-VPRAC2pdf/
• https://capricorn.bc.edu/bi204/wp-content/uploads/2015/08/Chapter-14-2015.pdf
• https://www.slideshare.net/rahemematthie/human-social-biology-sample-project-on-the-impact-of-heathpractices-on-the-environment
• https://www.creative-diagnostics.com/Sample-Gel-Preparation.htm

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• SDS-PAGE: Introduction, Principle, Working and Steps