Electrophoresis

Electrophoresis

Electrophoresis is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. This electrokinetic phenomenon was observed for the first time in 1807 by Ferdinand Frederic Reuss. It is the basis for a number of analytical techniques used in biochemistry for separating molecules by size, charge, or binding affinity.

Electrophoresis of positively charged particles (cations) is called cataphoresis, while electrophoresis of negatively charged particles (anions) is called anaphoresis.

 

Capillary electrophoresis

Capillary electrophoresis is a electrokinetic separation methods performed in submillimeter capillaries. Very often, CE refers to capillary zone electrophoresis (CZE), but other electrophoretic techniques including capillary gel electrophoresis (CGE), capillary isoelectric focusing (CIEF), capillary isotachophoresis and micellar electrokinetic chromatography (MEKC) belong also to this class of methods. In CE methods, analytes migrate through electrolyte solutions under the influence of an electric field. Analytes can be separated according to ionic mobility, additionally they may be concentrated by means of gradients in conductivity and pH.

CAP

The instrumentation needed to perform capillary electrophoresis is relatively simple. The system’s main components are a sample vial, source and destination vials, a capillary, electrodes, a high-voltage power supply, a detector  and a data output and handling device. The source vial, destination vial and capillary are filled with an electrolyte such as an aqueous buffer solution. To introduce the sample, the capillary inlet is placed into a vial containing the sample. Sample is introduced into the capillary via capillary action, pressure, or electrokinetically, and the capillary is then returned to the source vial. The migration of the analytes is initiated by an electric field that is applied between the source and destination vials and is supplied to the electrodes by the high-voltage power supply. In the most common mode of CE, all ions, positive or negative, are pulled through the capillary in the same direction by electroosmotic flow. The analytes separate as they migrate due to their electrophoretic mobility and are detected near the outlet end of the capillary. The output of the detector is sent to a data output and handling device such as an integrator or computer. The data is then displayed as an electropherogram, which reports detector response as a function of time.

PAGE

Polyacrylamide gel electrophoresis (PAGE), describes a technique widely used in biochemistry, forensics, genetics, molecular biology and biotechnology to separate biological macromolecules, usually proteins, according to their electrophoretic mobility. Mobility is a function of the length, conformation and charge of the molecule.

As with all forms of gel electrophoresis, molecules may be run in their native state, preserving the molecules higher-order structure, or a chemical denaturant may be added to remove this structure and turn the molecule into an unstructured linear chain whose mobility depends only on its length and mass-to-charge ratio. For proteins, sodium dodecyl sulfate (SDS) is an anionic detergent applied to protein sample to linearize proteins and to impart a negative charge to linearized proteins. This procedure is called SDS-PAGE. In most proteins, the binding of SDS to the polypeptide chain imparts an even distribution of charge per unit mass, thereby resulting in a fractionation by approximate size during electrophoresis.

Sample preparation

Samples may be any material containing proteins. These may be biologically derived, for example from prokaryotic or eukaryotic cells, tissues, viruses, environmental samples, or purified proteins. In the case of solid tissues or cells, these are often first broken down mechanically using a blender (for larger sample volumes), using a homogenizer (smaller volumes), by sonicator or by using cycling of high pressure, and a combination of biochemical and mechanical techniques – including various types of filtration and centrifugation – may be used to separate different cell compartments and organelles prior to electrophoresis. Synthetic biomolecules such as oligonucleotides may also be used as analytes.

The sample to analyze is optionally mixed with a chemical denaturant, usually SDS for proteins. 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. Heating the samples to at least 60°C further promotes denaturation.

Preparing acrylamide gels

The gels typically consist of acrylamide, bisacrylamide, the optional denaturant (SDS), and a buffer with an adjusted pH. The solution may be degassed under a vacuum to prevent the formation of air bubbles during polymerization. A source of free radicals and a stabilizer, such as ammonium persulfate and TEMED are added to initiate polymerization. The polymerization reaction creates a gel because of the added bisacrylamide, which can form cross-links between two polyacrylamide molecules. 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.

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  to migrate across the gel from the negative electrode (the cathode) 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; migration occurs more quickly at higher voltages, but these results are typically less accurate than at those at lower voltages. After the set amount of time, the biomolecules 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 R-250 or 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 the distance traveled relative to the marker.

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