Polyacrylamide Gel Electrophoresis (PAGE)
Polyacrylamide gel electrophoresis (PAGE) is a technique that allows the separation and analysis of proteins and nucleic acids based on their size and charge. PAGE can be performed under denaturing or native conditions, depending on the purpose of the experiment. Denaturing conditions disrupt the secondary and tertiary structure of the biomolecules, making them linear and uniformly charged. Native conditions preserve the natural conformation and charge of the biomolecules, allowing the separation based on both size and shape.
To perform PAGE, you will need the following materials and equipment:
- Acrylamide solutions: These are the main components of the polyacrylamide gel. You will need two different solutions: one for the resolving gel and one for the stacking gel. The resolving gel has a higher concentration of acrylamide and bisacrylamide than the stacking gel, and it is where the separation of proteins or nucleic acids occurs. The stacking gel has a lower concentration of acrylamide and bisacrylamide, and it is where the samples are concentrated before entering the resolving gel. The acrylamide solutions also contain a buffer (usually Tris-HCl) to maintain the pH of the gel, and a catalyst (usually ammonium persulfate) to initiate the polymerization reaction.
- Isopropanol or distilled water: These are used to create a smooth surface on top of the resolving gel while it is polymerizing. Isopropanol prevents oxygen from interfering with the polymerization reaction, while distilled water creates a layer of hydration that helps the stacking gel adhere to the resolving gel.
- Gel loading buffer: This is a solution that is added to the protein or nucleic acid samples before loading them onto the gel. The gel loading buffer contains a tracking dye (usually bromophenol blue) that allows you to monitor the progress of electrophoresis, a reducing agent (usually dithiothreitol or mercaptoethanol) that breaks disulfide bonds in proteins and prevents them from forming aggregates, and a denaturing agent (usually SDS or urea) that unfolds the proteins or nucleic acids and gives them a uniform negative charge.
- Running buffer: This is the solution that fills the electrophoresis chamber and surrounds the gel. The running buffer provides an electric field for the migration of proteins or nucleic acids through the gel, and also maintains the pH and ionic strength of the system. The running buffer usually contains Tris, glycine, and SDS for proteins, or Tris, borate, and EDTA for nucleic acids.
- Staining and destaining solutions: These are solutions that are used to visualize the proteins or nucleic acids after electrophoresis. The staining solution contains a dye that binds to the proteins or nucleic acids and makes them visible as bands on the gel. The destaining solution removes excess dye from the gel and enhances the contrast between stained and unstained regions. The most common staining solution for proteins is Coomassie Brilliant Blue, while for nucleic acids it is ethidium bromide. The most common destaining solution for both proteins and nucleic acids is water or a mixture of water and methanol or acetic acid.
- Protein samples: These are the biological materials that contain the proteins of interest. They can be obtained from various sources, such as cells, tissues, organs, fluids, etc. The protein samples need to be prepared before loading onto the gel by homogenizing, lysing, centrifuging, precipitating, concentrating, or purifying them as needed. The protein samples also need to be mixed with an appropriate volume of gel loading buffer before loading onto the gel.
- Molecular weight markers: These are known mixtures of proteins or nucleic acids with different molecular weights that are loaded onto separate lanes of the gel along with the samples. The molecular weight markers serve as references for estimating the molecular weight of unknown proteins or nucleic acids by comparing their migration distances on the gel. The molecular weight markers should have a similar composition and charge as the samples, and should cover a wide range of molecular weights that include those of the samples.
- Electrophoresis chamber and power supply: These are the devices that enable electrophoresis to take place. The electrophoresis chamber consists of a tank that holds the running buffer and two electrodes (anode and cathode) that generate an electric field across the gel. The power supply provides a constant voltage or current to the electrodes and allows you to control the parameters of electrophoresis, such as voltage, current, time, etc.
- Glass plates: These are thin rectangular sheets of glass that hold the polyacrylamide gel in place. You will need two glass plates: a short plate and a top plate. The short plate has a notch at one end that allows you to insert a comb to create wells in the stacking gel. The top plate has spacers on its sides that create a uniform thickness for the gel. The glass plates are clamped together with binder clips or screws to form a sandwich that contains the polyacrylamide gel.
- Casting frame: This is a metal frame that holds the glass plates together while pouring and polymerizing the polyacrylamide gel. The casting frame has screws or clamps that secure the glass plates in position and prevent them from leaking.
- Casting stand: This is a metal stand that supports the casting frame in an upright position while pouring and polymerizing the polyacrylamide gel. The casting stand has slots or grooves that fit the casting frame snugly and keep it stable.
- Combs: These are plastic or metal devices that create wells in the stacking gel where you can load your samples and molecular weight markers. The combs have teeth that correspond to the number and size of wells you want to create in your gel. The combs are inserted into the notch in the short plate before pouring the stacking gel, and removed after the stacking gel has polymerized.
These are all the requirements for performing PAGE. In the next section, we will discuss the steps involved in PAGE in more detail.
- Samples may be any material containing proteins or nucleic acids.
- The sample to analyze is optionally mixed with a chemical denaturant if so desired, usually SDS for proteins or urea for nucleic acids.
- SDS is an anionic detergent that denatures secondary and non–disulfide–linked tertiary structures, and additionally applies a negative charge to each protein in proportion to its mass. Urea breaks the hydrogen bonds between the base pairs of the nucleic acid, causing the constituent strands to separate. Heating the samples to at least 60 °C further promotes denaturation.
- A tracking dye may be added to the solution. This typically has a higher electrophoretic mobility than the analytes to allow the experimenter to track the progress of the solution through the gel during the electrophoretic run.
Preparation of polyacrylamide gel
- The gels typically consist of acrylamide, bisacrylamide, the optional denaturant (SDS or urea), and a buffer with an adjusted pH.
- The ratio of bisacrylamide to acrylamide can be varied for special purposes, but is generally about 1 part in 35. The acrylamide concentration of the gel can also be varied, generally in the range from 5% to 25%.
- Lower percentage gels are better for resolving very high molecular weight molecules, while much higher percentages of acrylamide are needed to resolve smaller proteins.
- Gels are usually polymerized between two glass plates in a gel caster, with a comb inserted at the top to create the sample wells.
- After the gel is polymerized the comb can be removed and the gel is ready for electrophoresis.
- Various buffer systems are used in PAGE depending on the nature of the sample and the experimental objective.
- The buffers used at the anode and cathode may be the same or different.
- An electric field is applied across the gel, causing the negatively charged proteins or nucleic acids to migrate across the gel away from the negative and towards the positive electrode (the anode).
- Depending on their size, each biomolecule moves differently through the gel matrix: small molecules more easily fit through the pores in the gel, while larger ones have more difficulty.
- The gel is run usually for a few hours, though this depends on the voltage applied across the gel.
- After the set amount of time, the biomolecules will have migrated different distances based on their size.
- Smaller biomolecules travel farther down the gel, while larger ones remain closer to the point of origin.
- Biomolecules may therefore be separated roughly according to size, which depends mainly on molecular weight under denaturing conditions, but also depends on higher-order conformation under native conditions.
- Following electrophoresis, the gel may be stained (for proteins, most commonly with Coomassie Brilliant Blue or autoradiography; for nucleic acids, ethidium bromide; or for either, silver stain), allowing visualization of the separated proteins, or processed further (e.g. Western blot).
- After staining, different species biomolecules appear as distinct bands within the gel.
- It is common to run molecular weight size markers of known molecular weight in a separate lane in the gel to calibrate the gel and determine the approximate molecular mass of unknown biomolecules by comparing their migration distance with that of a known molecular weight ladder (marker).
Polyacrylamide gel electrophoresis (PAGE) is a versatile and widely used technique for the separation and analysis of proteins and nucleic acids. Some of the applications of PAGE are:
- Measuring molecular weight: By comparing the migration distance of unknown samples with that of known molecular weight markers, the approximate molecular weight of proteins or nucleic acids can be estimated. This can help in identifying or characterizing the samples.
- Peptide mapping: By digesting a protein with a specific enzyme and separating the resulting peptides by PAGE, the peptide map or fingerprint of the protein can be obtained. This can provide information about the amino acid sequence, post-translational modifications, and structural features of the protein.
- Estimation of protein size: By using SDS-PAGE, which denatures and uniformly charges the proteins, the size of proteins can be estimated based on their electrophoretic mobility. This can help in distinguishing between different isoforms or variants of a protein.
- Determination of protein subunits or aggregation structures: By using native PAGE, which preserves the native conformation and interactions of proteins, the subunit composition or aggregation state of a protein can be determined. This can provide insights into the quaternary structure, oligomerization, or complex formation of proteins.
- Estimation of protein purity: By visualizing the protein bands after staining, the purity or contamination level of a protein sample can be assessed. This can help in evaluating the quality or yield of a protein purification process.
- Protein quantitation: By measuring the intensity or density of the protein bands after staining, the amount or concentration of a protein in a sample can be quantified. This can help in determining the stoichiometry or kinetics of a protein reaction or interaction.
- Monitoring protein integrity: By detecting any changes in the electrophoretic pattern or band intensity of a protein sample over time or under different conditions, the stability or degradation of a protein can be monitored. This can help in assessing the shelf-life or storage conditions of a protein product or reagent.
- Comparison of the polypeptide composition of different samples: By running multiple samples on the same gel and comparing their electrophoretic profiles, the similarities or differences in the polypeptide composition of different samples can be revealed. This can help in identifying or classifying different types or sources of samples, such as tissues, cells, organisms, etc.
- Analysis of the number and size of polypeptide subunits: By using SDS-PAGE under reducing or non-reducing conditions, which break or preserve the disulfide bonds between polypeptide chains, respectively, the number and size of polypeptide subunits within a protein can be analyzed. This can provide information about the primary structure, folding, or cross-linking of proteins.
- Post-electrophoresis applications: After separation by PAGE, the proteins or nucleic acids in the gel can be further processed for various purposes, such as Western blotting (for immunodetection), Southern blotting (for DNA hybridization), Northern blotting (for RNA hybridization), mass spectrometry (for identification or characterization), sequencing (for determining nucleotide or amino acid order), etc.
These are some examples of how PAGE can be applied to various fields and questions in biological research and biotechnology. However, this is not an exhaustive list and there may be other applications that are not mentioned here. PAGE is a powerful and flexible technique that can be adapted to suit different needs and objectives. 😊
Polyacrylamide gel electrophoresis (PAGE) is a widely used technique for separating proteins or nucleic acids based on their size and charge. Like any other technique, it has some advantages and disadvantages that should be considered before using it.
Some of the advantages of PAGE are:
- It has a stable chemically cross-linked gel that can withstand high voltages and temperatures without melting or breaking.
- It has a greater resolving power than agarose gel electrophoresis, meaning that it can separate molecules with small differences in size or charge more clearly and sharply.
- It can accommodate larger quantities of DNA without significant loss in resolution, which is useful for applications such as DNA sequencing or fingerprinting.
- The DNA recovered from polyacrylamide gels is extremely pure, as it does not contain any agarose or other contaminants that may interfere with downstream analysis or manipulation.
- The pore size of the polyacrylamide gels can be altered in an easy and controllable fashion by changing the concentrations of the two monomers, acrylamide and bisacrylamide. This allows for the optimization of the separation conditions for different types of molecules.
- It is good for separation of low molecular weight fragments, such as oligonucleotides or peptides, that may not be resolved well by agarose gel electrophoresis.
Some of the disadvantages of PAGE are:
- It is generally more difficult to prepare and handle, involving a longer time for preparation than agarose gels. It also requires special equipment and supplies, such as glass plates, casting frames, combs, and gel cassettes.
- It involves the use of toxic monomers, such as acrylamide and bisacrylamide, that can cause skin irritation, nerve damage, or cancer if inhaled or ingested. Therefore, proper safety precautions and protective equipment are necessary when working with these chemicals.
- The gels are tedious to prepare and often leak, especially if the gel cassette is not sealed properly or if air bubbles are trapped in the gel. This can result in uneven polymerization, distortion, or loss of the gel.
- A new gel has to be prepared for each experiment, as polyacrylamide gels cannot be reused or stored for long periods of time. This increases the cost and waste of the technique.
In conclusion, PAGE is a powerful and versatile technique for separating molecules based on their size and charge, but it also has some drawbacks that should be weighed against its benefits. Depending on the purpose and scope of the experiment, PAGE may or may not be the best choice among the available electrophoresis methods.
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