Polyacrylamide Gel Electrophoresis

• Since both agarose and polyacrylamide gels are porous in nature, electrophoresis via both gels is a widely used technique for separating, identifying, and purifying biopolymers.
• Through the polymerization of acrylamide using a cross-linking agent, often N, N’-methylenebisacrylamide, polyacrylamide gels are created that are chemically cross-linked gels.
• The process, a free radical polymerization, is typically carried out using N,N,N’,N’-tetramethylethylendiamine (TEMED) as the catalyst and ammonium persulfate as the initiator.
• In biochemistry, forensic chemistry, genetics, molecular biology, and biotechnology, polyacrylamide gel electrophoresis (PAGE) is a regularly used technique for separating biological macromolecules based on their electrophoretic mobility.
• The sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), which is primarily used for the separation of proteins, is the method of polyacrylamide gel electrophoresis that is most often used.

Principle of Polyacrylamide Gel Electrophoresis (PAGE)

An analytical technique, called SDS-PAGE (Polyacrylamide Gel Electrophoresis) according to their diameters, separates the components of a protein mixture.

According to this approach, a charged molecule will migrate to an electrode having the opposite sign. Because the movement of a substance in a gel is dependent on both size and charge, standard electrophoresis techniques cannot be utilised to determine the molecular weight of biomolecules.

In order to overcome this, the biological samples should be treated to provide them with a homogenous charge; afterwards, the electrophoretic motility is mainly influenced by size. To do this, different protein molecules of different sizes and shapes must be denatured (carried out using SDS) in order for the proteins to shed their secondary, tertiary, or quaternary structures.

While injected onto a gel and subjected to an electric field, the negatively charged proteins getting covered by SDS will move toward the anode (positively charged electrode). This migration is based on a molecular sieve action, which separates the proteins depending on size. A protein’s size may be measured by matching its migrating range to that of a recognised molecular weight ladder after the protein has been stained (protein-specifically) and visualised (marker).

Requirements for Polyacrylamide Gel Electrophoresis (PAGE)

• Acrylamide solutions (for resolving and stacking gels).
• Isopropanol and distilled water.
• Gel loading buffer.
• Running buffer
• Solutions for staining and destaining
• Samples of proteins
• markers of molecular weight.

The equipment and supplies necessary for conducting SDS-PAGE include:

• Power source and electrophoresis chamber.
• Glass plates (a short and a top plate).
• Frame for casting.
• Stand for casting.
• Combs

Steps Involved in Polyacrylamide Gel Electrophoresis (PAGE)

1. Preparing a sample

• As a sample, any material containing proteins or nucleic acids is suitable.
• If desired, a chemical denaturant, typically SDS for proteins or urea for nucleic acids, is added to the sample before analysis.
• SDS is an anionic detergent which inactivates tertiary, secondary, and non-disulfide-linked structures as well as negatively charges every protein according to its weight. As a consequence of urea’s breakdown of the hydrogen bonds binding the base pairs together, the nucleic acid strands anneal.The denaturation process is hastened via heating the specimens to a minimum of 60 degrees Celsius.
• The fluid may be infused with tracking dye. Typically, this has a larger electrophoretic motion than the analytes so that the experimenter may observe the circulation of the solution through the gel during the electrophoretic run.

2. Preparation of polyacrylamide gel

• Acrylamide, bisacrylamide, the optional denaturant (SDS, or urea), and a buffer with a pH-adjusted pH make up most gels.
• The ratio of bisacrylamide to acrylamide may be altered for specific applications, but it is normally 1 part in 35. The acrylamide content of the gel may also be modified; it typically ranges from 5 to 25 percent.
• Gels with a lower percentage of acrylamide are better for resolving molecules with an exceptionally high molecular weight, despite the fact that substantially higher concentrations of acrylamide are needed to separate smaller proteins.
• In a gel caster, gels are typically polymerized between two glass plates, with the sample wells being created by inserting a comb at the top.
• The comb may be removed from the gel when it has polymerized, and it is then ready for electrophoresis.

3. Electrophoresis

• Depending on the sample’s characteristics and the intended purpose of the experiment, several buffer systems are employed in PAGE.
• The buffers used for the anode and cathode may be similar or different.
• When an electric field is generated, negatively charged proteins or nucleic acids move through the gel, away from the negative electrode and in the direction of the positive electrode (the anode).
• Based on its size, every biomolecule acts uniquely in the gel matrix; smaller molecules are more easily able to flow through the gel’s pores.
• Depending on the voltage put across the gel, the gel is typically operated for a few hours.
• Depending on their size, the biomolecules will have moved a variety of lengths after the predetermined period of time.
• Larger biomolecules stay near to their site of origin, whereas smaller ones go farther down the gel.
• Therefore, biomolecules may be generally classified based on size, which under denaturing circumstances is primarily determined by molecular weight but also by conformation of a higher order under natural circumstances.

4. Detection

• After electrophoresis, the gel might undergo further processing or be coloured (usually with Coomassie Brilliant Blue or autoradiography for proteins; ethidium bromide for nucleic acids; or silver stain for either). This permits the separation of proteins to be seen (e.g., Western blot).
• Following staining, the gel shows discrete bands representing the many types of biomolecules.
• In order to standardise the gel and determine the average molecular mass of unidentified biomolecules by calculating the range of movement compared to the marker, it is customary to operate a molecular weight-size marker of recognised molecular weight in a different lane of the gel.

Applications of Polyacrylamide Gel Electrophoresis (PAGE)

• the molecular weight measurement.
• Mapping peptides.
• Measurement of protein size.
• Figuring out the architecture of protein aggregations or components
• Purity of proteins is estimated.
• Protein measurement.
• Keeping an eye on protein integrity.
• Analyzing and contrasting the polypeptide content of various samples
• Examination of the quantity and dimensions of polypeptide subunits
• Applications performed after electrophoresis, such as Western blotting.
• Coomassie G-250 Staining of Proteins in Gels Without Organic Solvent and Acetic Acid
• Using commercial cassettes to pour and run a protein gel
• CyDye DIGE Fluor Minimal Selective Labelling of Cell-surface ProteinsDyes
• Protein ubiquitination is detected.

Advantages of Polyacrylamide Gel Electrophoresis (PAGE)

• Chemically cross-linked gel that is stable
• increased resolving capacity (Sharp bands)
• may contain more DNA without suffering a severe resolution loss
• Extremely pure DNA was retrieved from polyacrylamide gels.
• By varying the amounts of the two monomers, it is simple and manageable to change the pore size of polyacrylamide gels.
• Separating low molecular weight fragments well

Disadvantages of Polyacrylamide Gel Electrophoresis (PAGE)

• It often takes longer to prepare than agarose gels and is more challenging to handle.
• Harmful monomers
• Gels are difficult to make and often leak.
• You need fresh gel for every experiment. chemically cross-linked gel that is stable

References

1. http://elte.prompt.hu/sites/default/files/tananyagok/IntroductionToPracticalBiochemistry/ch07s03.html
2. https://www.wou.edu/las/physci/ch462/Gel%20Electrophoresis.pdf
3. https://www.slideshare.net/mbn1994/introduction-principle-instrumentation-and-applications-of-sdspage-55728195
4. https://en.wikipedia.org/wiki/Polyacrylamide_gel_electrophoresis https://msu.edu/course/css/451/Lecture/PT-electrophoresis%20(2009).pdf
5. http://library.umac.mo/ebooks/b28050459.pdf
6. http://vlab.amrita.edu/?sub=3&brch=186&sim=319&cnt=1