Electrophoresis is an analytical technique for the analysis of macromolecules like proteins and nucleic acids. This technique was discovered and first used in 1937 by a Swedish biochemist Arne Tiselius . The electrophoretic effect is based on the theory of Debye - Huckel - Onsager where this theory of electrolytic dissociation accept the fact that charged particles move up under the influence of electrostatic forces to an electrode of opposite charge is applied when a potential difference in a solution containing electrolytes.
Electrophoresis is the migration of a charged molecule under the influence of an electric field . The electrophoretic mobility is given by :
μ = v / E = Z / f
where :
The electrophoretic mobility ( μ ) is the ratio between the speed ( v) the macromolecule and the electric potential ( E) that moves the macromolecule or the ratio of the net charge (Z) and the friction coefficient (f).
Media for electrophoresis
The media commonly used for electrophoresis are polyacrylamide gels for proteins and nucleic acids in agarose by virtue of these polymers function as a molecular sieve , or separate species due to its size and molecular weight , respectively , inhibits propagation of heat due to the friction caused by the migration and application of electric field.
Polyacrylamide gels are commonly used for protein separation by virtue of being chemically inert , easy staining with silver nitrate and Coomassie blue dye (dyes such as agarose stain completely prevented the identification of species in the electrophoretogram ) , the pores are easyly adjustable through control of acrylamide and bis- acrylamide are polymers that form the gel.
The agarose gels are used for macmoléculas with a molecular weight greater tha...
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... focusing is placed horizontally on an SDS gel , the electric field is applied and the protein macromolecules are separated acrodo with the isoelectric point and in terms of its molecular mass , providing a electrophoretogram that enables viewing and identification of many proteins at once .
Two-dimensional electrophoresis or 2D
This technique offers better resolution , or a higher level of detail at the end of which combines electrophoretogram isoelectric focusing and SDS gel electrophoresis . According to the scheme below :
In two-dimensional electrophoresis gel of isoelectric focusing is placed horizontally on an SDS gel , the electric field is applied and the protein macromolecules are separated acrodo with the isoelectric point and in terms of its molecular mass , providing a electrophoretogram that enables viewing and identification of many proteins at once .
Digestion of the haemolytic and non-haemolytic cells allowed for easier identification of fragments during electrophoresis analysis. Lane 12 in figure 3 show the size markers of SPP1 digested with EcoR1 while lanes 6 and 7 show samples of pK184hlyA and pBluescript digested with EcoR1 and Pst1. Lane 4 was loaded with plasmid DNA from haemolytic cells digested with EcoR1 and Pst1 while lane 5 was loaded with EcoR1 and Pst1 digested DNA from non-haemolytic cells. There was a lack of technical success in both lanes due to no bands appearing in lane 4 and only a single band appearing in lane 5. Theoretically, two bands should appear in both lanes after successful to allow for fragment identification. A possible explanation for the single, large fragment in lane 5 is that successful digestion did not take place and the plasmid was only cut at one restriction site leaving a large linear fragment of plasmid DNA. The absence of bands in lane 4 could be because there was not enough plasmid loaded into the lane. Another possibility could be that low plasmid yield as obtained when eluting the experimental samples in order to purify it. Lanes 8 and 9 belonged to another group and show technical success as two bands were present in both the haemolytic (lane 8) and non-haemolytic (lane 9) lanes. If the
electrophoresis gel and the device was turned on. Using the methods discussed above we found
The purpose of this experiment was to discover the specificity of the enzyme lactase to a spec...
During this time, it could only be used in a lab with semi-intense supervision. Now, fast forward a few decades and there are D.I.Y. at home kits. The process of Electrophoresis starts with an electric current being run through a gel containing the molecules of interest. The molecules will then travel through the gel in different directions and speeds, based on their size and charge, allowing them to be separated from each other. Dyes, fluorescent tags, and radioactive labels can all enable the molecules on the gel to be seen after they have been separated. Because of these identification markers, they appear as a band across the top of the gel. Electrophoresis can be used for many different things. It is used to identify and study DNA or DNA fragments, and helps us to better understand the molecular components of both living and deceased organisms. Electrophoresis can also be used to test for genes related to specific diseases and life altering diagnoses such as Multiple Sclerosis, Down’s Syndrome, kidney disease, and some types of cancer. Electrophoresis also plays a major role in the testing of antibiotics. It can be used to determine the purity and concentration of one specific type of antibiotic or several general antibiotics at a time. Electrophoresis is also extremely useful in the creation and testing of
Gel filtration is one of the many methods that can be used for purifying proteins. Gel filtration, also known as size-exclusion chromatography, separates proteins based on size. There are pros and cons for this methods. One of the pros is that fragile proteins will not or hardly get damaged when going through the chromatography column. One of the cons is the results are not very accurate due to the proteins’ inability to adhere to the column at times.
This cell membrane plays an important part in Diffusion. Cell membrane and Diffusion Diffusion is the movement of the molecules of gas or liquids from a higher concentrated region to a lower concentration through the partially permeable cell membrane along a concentraion gradient. This explanation is in the diagram shown below: [IMAGE] Turgor When a plant cell is placed in a dilute solution or a less concentrated solution then the water particles pass through the partially permeable membrane and fill the cell up with water. The cell then becomes Turgor or hard. An example of this is a strong well-watered plant.
This occurs when special carrier proteins carry solutes dissolved in the water across the membrane by using active transport. When the concentration gradient can not allow travel from one side of the membrane to the other fast enough for the cell’s nutritional needs, then facilitated diffusion is used. The transport protein is specialized for the solute it is carrying, just as enzymes are specialized for their substrate. The transport protein can be
When a cell membrane is said to be selectively permeable, it means that the cell membrane controls what substances pass in and out through the membrane. This characteristic of cell membranes plays a great role in passive transport. Passive transport is the movement of substances across the cell membrane without any input of energy by the cell. The energy for passive transport comes entirely from kinetic energy that the molecules have. The simplest type of passive transport is diffusion, which is the movement of molecules from an area of high concentration to an area of lower concentration. Diffusion
When a positive and a negative electrode are placed in a solution containing ions, and an electric potential is applied to the electrodes, the positively charged ions move towards the negative electrode, and the negatively charged ions to the positive electrode. As a result, an electric current flows between the electrodes. The strength of the current depends on the electric potential between the electrodes and the concentration of ions in the solution. Ionization is the formation of electrically charges atoms or molecules.
The current moves the molecules towards the cathode or anode. The speed of the moving molecules depends on the size, shape, and charge. The properties of the gel will definitely affect the movement. Small molecules are expected to move easily and faster through the pores. Materials and Methods: Experiment: 1st step to make the gel: pour distilled water and agarose in a beaker.
The purpose of this lab was to see firsthand the diffusion of a substance across a selectively permeable membrane. Diffusion is the movement of molecules from an area of high concentration to an area of lower concentration until both concentrations are equal, or as you could more professionally call it, equilibrium. This concept is one that we have been studying in depth currently in Biology class.
Gel electrophoresis is used in a variety of settings, particularly in molecular biology. Besides being used to separate nucleic acids, such as DNA and RNA, gel electrophoresis is also employed to divide proteins (Gel Electrophoresis). According to research, electrophoresis is applied for the following reasons, "To get a DNA fingerprint for forensic pur...
Leboffe, M. J., & Pierce, B. E. (2010). Microbiology: Laboratory Theory and Application, Third Edition 3rd Edition (3rd Ed.). Morton Publishing
Staining is used in a variety of ways in order to color the background of a cell, discern types of cells and to discern structures of a cell. A differential stain is when multiple dyes are used to stain a cell that take advantage of chemical differences in a cell. Gram staining is a type of differential stain that works by distinguishing gram positive and gram negative cells by coloring them violet or red, respectively. Gram positive cells contain a thick cell wall of peptidoglycan and a single membrane. Gram negative cells contain a thin cell wall which is located between two membrane layers. There are four reagents used in gram staining which include crystal violet, iodine, ethanol and basic fuchsin. Crystal violet is a primary methyl
The next step is to purify the protein Ara h1 using DEAE Fast Flow and high performance liquid chromatography. The purity of Ara h1 was measured by SDS-PAGE gel stained with Coomassie Brilliant Blue.