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Electrophoresis



Electrophoresis is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field.[1][2][3][4][5][6][7] Electrophoresis of positively charged particles (cations) is sometimes called cataphoresis, while electrophoresis of negatively charged particles (anions) is sometimes called anaphoresis.




Electrophoresis



The electrokinetic phenomenon of electrophoresis was observed for the first time in 1807 by Russian professors Peter Ivanovich Strakhov and Ferdinand Frederic Reuss at Moscow University,[8] who noticed that the application of a constant electric field caused clay particles dispersed in water to migrate. It is ultimately caused by the presence of a charged interface between the particle surface and the surrounding fluid. It is the basis for analytical techniques used in chemistry for separating molecules by size, charge, or binding affinity.


This model of "thin double layer" offers tremendous simplifications not only for electrophoresis theory but for many other electrokinetic theories. This model is valid for most aqueous systems, where the Debye length is usually only a few nanometers. It only breaks for nano-colloids in solution with ionic strength close to water.


Immunoelectrophoresis and immunofixation electrophoresis: Initially, proteins are separated on the agarose gel. Wells are created after separation, and specific antibodies against molecules of interest are added. Bands of precipitation due to antigen-antibody reaction are formed, which signifies the presence of a specific protein in the sample. It is used to identify the abnormal elevation of gamma globulin fractions and free light chains in patients with suspected monoclonal or polyclonal gammopathy.[11]


High voltage electrophoresis: Relatively higher voltage (400 to 2000 Volts) is used instead of 250 Volts for separation. It provides very fast separation with good resolution and relatively less diffusion. It is used for the separation of proteins, hemoglobin, and nucleotides.[5]


Pulse field electrophoresis: Separation of long nucleotide fragments with good resolution is difficult with conventional electrophoresis. In pulse field electrophoresis, the current is passed in two different directions alternatively, which leads to the movement of fragments in two directions, giving good separation with optimum resolution.[12]


Capillary electrophoresis: Very small diameter capillary tube filled with buffer solution, ampholytes, or gel is used as a support medium. Due to the availability of a higher surface area for heat dissipation, very high voltage can be applied for speedy separation and better resolution. Separated fractions can be quantified simultaneously as they pass through the detector during the electrophoretic run.[13]


2-dimensional (2D) electrophoresis: Initially, isoelectric focusing (IEF) is done to separate the analytes based on their isoelectric pH. The gel containing separated analytes is then subjected to SDS-polyacrylamide gel electrophoresis in the direction of 90 degrees to IEF run. Molecules having similar molecular weight can be separated via this method due to differences in their isoelectric pH.[5][10]


Depending on the type of electrophoresis and disease involved, specimen requirement and processing vary. Serum, plasma, whole blood, and hemolysate are the most commonly used biological specimens in diagnostic laboratory setups.


The major interfering factors are heat, non-specific adsorptive groups on the support medium, and electroendosmosis. As the current and duration of electrophoresis increase, the gel's temperature increases due to heat dissipation. This will increase the random motion of the molecules in the medium, reducing the sharpness/resolution of the separated bands.


After the electrophoresis run is over, the gel is stained for the analyte of interest. After incubation with the staining solution, excess of the stain is removed by treating the gel with the de-staining solution.


The electrophoresis technique can also be used to identify abnormally elevated or decreased enzyme isoforms. Various clinical conditions show specific enzyme patterns based on tissue/organ involvement which can help the clinician to diagnose and develop a treatment plan for the case. The following table shows the tissue origin of different isoforms of some plasma non-functional enzymes of clinical significance.


The utility of electrophoresis is not limited to diagnostics. It is widely utilized for research in genomics and proteomics. Restriction fragment length polymorphism (RFLP), nucleotide sequencing, next generation sequencing, southern blotting, western blotting, etc., are examples of a few techniques having electrophoresis as one of the steps in it. DNA fingerprinting is the technique utilized by forensic experts to compare DNA acquired from the crime scene and the DNA of the suspects and/or victims. DNA fingerprinting is also utilized for confirmation of the biological parents of a child in case of a dispute.[26]


Caution is necessary when preparing electrophoresis gel, buffer preparation, setting up apparatus, running of electrophoresis, staining, and visualization of the analyte. Monomers used in polyacrylamide gel preparation are carcinogenic. The catalyst used in polyacrylamide gel preparation can cause free radical-related damage to the skin if contacted. None of the solutions should be mouth pipetted. Barbital buffer containing sodium barbiturate is a known central nervous system depressant. Ethidium bromide used in nucleic acid staining is a known carcinogen. Direct exposure of the eye to ultraviolet rays during the visualization of the gel can cause severe damage to the eye.[27]


The diagnosis of the medical condition via electrophoresis is optimally performed and used with an interprofessional team that includes internal medicine, biochemistry, and laboratory medicine experts, along with nurses and laboratory technicians.


A technique has been developed for the separation of proteins by two-dimensional polyacrylamide gel electrophoresis. Due to its resolution and sensitivity, this technique is a powerful tool for the analysis and detection of proteins from complex biological sources. Proteins are separated according to isoelectric point by isoelectric focusing in the first dimension, and according to molecular weight by sodium dodecyl sulfate electrophoresis in the second dimension. Since these two parameters are unrelated, it is possible to obtain an almost uniform distribution of protein spots across a two-diminsional gel. This technique has resolved 1100 different components from Escherichia coli and should be capable of resolving a maximum of 5000 proteins. A protein containing as little as one disintegration per min of either 14C or 35S can be detected by autoradiography. A protein which constitutes 10 minus 4 to 10 minus 5% of the total protein can be detected and quantified by autoradiography. The reproducibility of the separation is sufficient to permit each spot on one separation to be matched with a spot on a different separation. This technique provides a method for estimation (at the described sensitivities) of the number of proteins made by any biological system. This system can resolve proteins differing in a single charge and consequently can be used in the analysis of in vivo modifications resulting in a change in charge. Proteins whose charge is changed by missense mutations can be identified. A detailed description of the methods as well as the characteristics of this system are presented.


Pulsed-field gel electrophoresis (PFGE) is a laboratory technique used by scientists to produce a DNA fingerprint for a bacterial isolate. A bacterial isolate is a group of the same type of bacteria. PulseNet investigates bacterial isolates from sick people, contaminated food, and the places where food is produced.


The DNA fragments produce a DNA fingerprint with a specific pattern. The figure shows an example of an agarose gel where each lane represents a DNA fingerprint or pattern. PFGE is different from conventional DNA electrophoresis because PFGE can separate very large fragments to generate a fingerprint by constantly changing the direction of the electric field.


We describe a modification of two-dimensional (2-D) polyacrylamide gel electrophoresis that requires only a single gel to reproducibly detect differences between two protein samples. This was accomplished by fluorescently tagging the two samples with two different dyes, running them on the same 2-D gel, post-run fluorescence imaging of the gel into two images, and superimposing the images. The amine reactive dyes were designed to insure that proteins common to both samples have the same relative mobility regardless of the dye used to tag them. Thus, this technique, called difference gel electrophoresis (DIGE), circumvents the need to compare several 2-D gels. DIGE is reproducible, sensitive, and can detect an exogenous difference between two Drosophila embryo extracts at nanogram levels. Moreover, an inducible protein from E. coli was detected after 15 min of induction and identified using DIGE preparatively.


Figure 5. An image of a gel post electrophoresis. EtBr was added to the gel before electrophoresis to a final concentration of 0.5 μg/ml, followed by separation at 100 V for 1 hour. The gel was exposed to uv light and the picture taken with a gel documentation system.


EtBr is the most common reagent used to stain DNA in agarose gels10. When exposed to uv light, electrons in the aromatic ring of the ethidium molecule are activated, which leads to the release of energy (light) as the electrons return to ground state. EtBr works by intercalating itself in the DNA molecule in a concentration dependent manner. This allows for an estimation of the amount of DNA in any particular DNA band based on its intensity. Because of its positive charge, the use of EtBr reduces the DNA migration rate by 15%. EtBr is a suspect mutagen and carcinogen, therefore one must exercise care when handling agarose gels containing it. In addition, EtBr is considered a hazardous waste and must be disposed of appropriately. Alternative stains for DNA in agarose gels include SYBR Gold, SYBR green, Crystal Violet and Methyl Blue. Of these, Methyl Blue and Crystal Violet do not require exposure of the gel to uv light for visualization of DNA bands, thereby reducing the probability of mutation if recovery of the DNA fragment from the gel is desired. However, their sensitivities are lower than that of EtBr. SYBR gold and SYBR green are both highly sensitive, uv dependent dyes with lower toxicity than EtBr, but they are considerably more expensive. Moreover, all of the alternative dyes either cannot be or do not work well when added directly to the gel, therefore the gel will have to be post stained after electrophoresis. Because of cost, ease of use, and sensitivity, EtBr still remains the dye of choice for many researchers. However, in certain situations, such as when hazardous waste disposal is difficult or when young students are performing an experiment, a less toxic dye may be preferred. 041b061a72


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