Electrophoresis with agarose or other polyacrylamide gels is an established method to separate the biopolymers, determine their identity and purify them because both of these gels have a porous the natural world. Polyacrylamide gels are chemically cross-linked gels formed by the polymerization of acrylamide with a cross-linking agent, usually N,N’-methylenebisacrylamide.
The reaction is a free radical polymerization, usually carried out with ammonium persulfate as the initiator and N,N,N’,N’-tetramethylethylendiamine (TEMED) as the catalyst. PAGE, also known as polyacrylamide gel electrophoresis (PAGE) can be described as a method extensively used for biochemistry,forensic chemical and genetics, as well as biotechnology, and molecular biology to identify biological macromolecules generally nucleic acids or proteins by the electrophoretic properties of their molecules.
The most widely used type of electrophoresis on polyacrylamide gels is Sodium dodecylsuplhate Polyacrylamide electrophoresis (SDS PAGE) which is used to separate proteins.
Principle of Polyacrylamide Gel Electrophoresis (PAGE)
SDS-PAGE (Polyacrylamide Gel Electrophoresis) is an analytical procedure that is used to identify the components of a protein mix according to their dimensions.
The technique is based on the concept that charged molecules move through an electric field to an electrode that has a sign opposite to it. The general electrophoresis methods cannot be employed in determining molecular mass of biological molecules as the speed of movement of a compound within the gel is dependent on the size and charge of the substance.
To get around this issue, the biological samples have to be treated in a way that they receive a uniform charge. the electrophoretic mobility will depend on the size. To overcome this, different proteins that have different sizes and shapes must be denatured (done by using SDS) in order that proteins are stripped of the secondary or tertiary, or the quaternary structure .The proteins that are coated by SDS are charged negatively and, when loaded onto a gel and put within an electric field they will move toward the electrode (positively positively charged electrode) is separated by a molecular Sieving effect that is based on the size. After the visualization using staining (protein-specific) method The size of a protein could be estimated by comparing the movement distance to that of the molecular weight ladder (marker).
Requirements for Polyacrylamide Gel Electrophoresis (PAGE)
- Acrylamide solutions (for resolving & stacking gels).
- Isopropanol / distilled water.
- Gel loading buffer.
- Running buffer.
- Staining, destaining solutions.
- Protein samples
- Molecular weight markers.
Other materials required for conducting SDS-PAGE includes:
- An electrophoresis chamber and power supply.
- Glass plates (a short and a top plate).
- Casting frame
- Casting stand
Steps Involved in Polyacrylamide Gel Electrophoresis (PAGE)
Step 1: Sample preparation
- The samples could be any substance that contains nucleic acids or proteins.
- The sample to be analyzed can be mixed with a chemical denaturant , if you wish to, typically SDS for proteins, or urea for nucleic acids.
- SDS is an anionic detergent that destroys secondary and non-disulfide linked tertiary structures in addition to imposing the negative charge to each proteins in relation to the mass. Urea breaks the hydrogen bonds that connect the bases of the nucleic acids, which causes the strands of protein to become annealable. The heating of the samples up to 60 degC will further accelerate denaturation.
- A tracking dye could include in the mix. This usually has a higher electrophoretic mobility than analytes that allow the scientist to observe the progression of the solution’s passage through the gel during the electrophoretic test.
Step 2: Preparation of polyacrylamide gel
- The gels usually comprise of bisacrylamide and acrylamide. the denaturant that is optional (SDS or Urea) and the buffer has an adjusted pH.
- The proportion of bisacrylamide to the acrylamide ratio can be altered depending on the purpose however it’s generally around 1 part per 35. The concentration of acrylamide in the gel may also be different, usually within the range of 5 to 25 percentage.
- Gels with lower percentages work better for the resolution of very high molecular weight molecules. On the other hand, more acrylamide-rich gels are required to dissect smaller proteins.
- Gels are generally made of two glass plates inside a gel caster. an insertion of a comb at the top of the comb to form the test wells.
- After the gel has polymerized the comb is removed after which the gel will be now ready to be electrophoresis.
Step 3: Electrophoresis
- Different buffer systems are employed in PAGE, based on the characteristics of the specimen and the objective of the study.
- The buffers that are used at the cathode and anode could be the same or different.
- A field of electricity is applied to the gel, causing positively charged protein or acid to move over the gel the negative electrode and toward an electrode that is positive (the the anode).
- In accordance with their size every biomolecule is different in its movement through the gel matrix. Small molecules can easily pass through the pores of the gel, while bigger ones face more trouble.
- The gel is typically run for a couple of hours but this is dependent on the voltage that is applied to the gel.
- After the specified duration, the biomolecules will have moved over various distances depending on the dimensions.
- Biomolecules that are smaller travel further through the gel, whereas larger biomolecules remain nearer to their point of origin.
- Biomolecules can therefore be classified by size which is primarily based on the molecular weight in denaturing situations, and also on higher-order conformation in native conditions.
Step 4: Detection
- After electrophoresis, the gel could stain (for proteins, typically using Coomassie Brilliant Blue or autoradiography or for nucleic acid, Ethidium Bromide or, for both silver stain) that allows for the visualisation of the protein fragments or further processed (e.g. Western Blot).
- After staining, different species of biomolecules show up as distinct bands in the gel.
- It is typical to use a molecular weight measurement marker of known molecular weight in a different channel in the gel to calibrate gel and estimate the molecular mass of biomolecules that are not known by measuring the distance traveled in relation with the marker.
Applications of Native Polyacrylamide Gel Electrophoresis (PAGE)
- Measurement of the molecular mass.
- Peptide mapping.
- Estimation of size of protein.
- Identification of protein subunits or aggregation patterns.
- Assessment of protein purity.
- Protein quantitation.
- Monitoring protein integrity.
- Comparative analysis of the polypeptide composition of various samples.
- Analyzing the size and number of subunits made up of polypeptides.
- Post-electrophoresis procedures, like Western Blotting.
- Staining of Proteins Gels with Coomassie G250 but not Organic Solvent and Acetic Acid.
- Pouring and running a Protein Gel using Commercial Cassettes.
- The selective labelling of cell-surface proteins with CyDye DIGE Small-scale Fluor Dyes.
- Detection of Protein Ubiquitination.
Advantages of Polyacrylamide Gel Electrophoresis (PAGE)
- Stable, chemically cross-linked gel that is stable and stable.
- Greater resolution capacity (Sharp bands)
- can accommodate greater quantities of DNA with no significant loss in resolution
- The DNA that is extracted from polyacrylamide gels are extremely pure
- The pores of polyacrylamide gels is adjustable in a simple and manageable manner by altering the concentrations of two monomers.
- It is useful for separating smaller molecular weight pieces
Disadvantages of Polyacrylamide Gel Electrophoresis (PAGE)
- The process is typically more difficult to handle and prepare taking longer to prepare over agarose gels.
- Monomers that are toxic
- It is a pain to make gels and are often leaking.
- You need a new gel for every experiment Stable cross-linked chemically