Immunology Practical: Enzyme Linked Immunosorbent Assay (ELISA)
Immunology Practical: Enzyme Linked Immunosorbent Assay (ELISA) Aim To acquire understanding and knowledge to determine the antibody titre of the Rabbit Anti-Ferritin Antibody using the Enzyme Linked Immunosorbent Assay (ELISA) Introduction ELISA is a rapid immunochemical assay that involves an enzyme (a protein that catalyzes a biochemical reaction). It also involves an antibody or antigen (immunologic molecules). The ELISA is a fundamental tool of clinical immunology, and is used as an initial screen for pathogens detection. Based on the principle of antibody-antibody interaction, in this assay you can easy visualisation the results and can be completed without the use of radioactive materials. The ELISA technique is the first and most basic assay to determine if an individual is positive for a selected pathogen, such as HIV. The applications of immunoassays are extended to other fields such as infectious diseases, autoimmune diseases, cancer, degenerative diseases, haematology and pharmacology. Applications have not been confined to human health care. Many applications have been described in veterinary medicine, agriculture, environmental health and the food industry. The assay is performed in a microtitre plate (plastic) which contains an 8 x 12 matrix of 96 wells. They also called indirect ELISA or sandwich ELISA. Microtitre plates are coated with antigen.
The solubilisation and purification of an intrinsic membrane protein presents problems distinct from those encountered in purifying a conventional soluble protein. Discuss this statement.
The solubilisation and purification of an intrinsic membrane protein presents problems distinct from those encountered in purifying a conventional soluble protein. Discuss this statement. Word count: 1860 In order to answer the question, this essay will first describe how soluble proteins are purified. It will then describe the process of solubilising an integral membrane protein specifically, and demonstrate differences between the two processes. There are several methods for the purification of proteins in aqueous solution. Since these methods discriminate based on one characteristic that may be shared by several proteins, it is almost always necessary to use multiple methods to purify a protein from its cellular environment. First, the cell must be homogenised in order to make all the proteins within available. In theory, this presents a problem since proteins are mixed with proteases, and could be degraded. In practice vacuoles form spontaneously and quickly to mitigate this effect so it is not a problem that has to be contended with. After homogenisation, there are several chromatographic methods available to purify proteins completely. Size exclusion chromatography separates proteins based on molecular weight, as smaller proteins are retarded by the resin and so take longer to flow through. Ion exchange chromatography involves charged resin which binds charged amino
The structure and function of membranes, with reference to membrane constituents and different types of membrane.
The structure and function of membranes, with reference to membrane constituents and different types of membrane. Membranes are essential for the compartmentalisation of a cell and therefore it is clear that the cell would struggle to develop any complexity without them. Although there are many different uses for membranes, especially in the eukaryotic cell, they serve a very important energetic purpose. For instance, they can retain a concentration gradient of, for example, Hydrogen ion, which means that the cell can use membranes not only for compartmentalising reactions, but also for compartmentalisation of energy and preventing diffusion. Therefore, without a complex system of membranes, the eukaryotic cell simply would not be able to carry out as many functions as it does. The prokaryotic cell differs in the sense that it does not possess membrane-bound organelles but still has an outer cell membrane. This has meant that it cannot carry out as many functions as the eukaryotic cells but its simplicity means that reproduction is far simpler. Therefore it is the membranes of the eukaryotic cell which are of more interest The basic constituents of all membranes are phospholipids, although there are different types of lipid present that I shall discuss later. All these molecules are amphipathic and therefore they have a polar 'head' and a hydrophobic 'tail'. They are based
Macromolecular composition of a liver cell
Macromolecular Composition of the Liver cell Abstract A liver cell is to be homogenised and fractionated into a nuclei rich sediment and a nuclei free supernatant using centrifugation. After treatment with perchloric acid the samples are centrifuged producing supernatants containing glycogen, and these are decanted and stored. The sediments are washed, then treated with KOH and perchloric acid and centrifuged again. This supernatant contains ribonucleotides and it is also stored. The remaining precipitates are suspended in KOH and incubated to ensure it is fully dissolved. The addition of various reagents to each of the supernatants and suspended sediments will allow for an examination of the distribution of RNA, DNA, glycogen and protein, and for an explanation of why this is so. Introduction For supernatants to be produced for examination of this kind, the liver cells must be fractionated to allow specific organelles and molecules to be collected. This is done through homogenisation and differential centrifugation. During homogenisation citric acid is added and in put in a pre-cooled homogeniser; liver is easily broken up. It would be relatively much more difficult to homogenise a plant cell due to the presence of a cell wall, an outer layer that maintains cell shape and is made of cellulose, other polysaccharides and protein (Campbell and Reece, 2005). A centrifuge
Transposon as tools for genetic engineering
Genomics Summer Semester 2011 Evaluation Essay Dmytro Dvornikov Genomics Summer Semester 2011. Evaluation essay Transposons have been widely used as genetic tools to study many model systems. However only recently have they been used in mammals. Why? Discuss the recent applications of transposition to mammalian systems and consider the advantages and disadvantages of transposition compared to other genetic methods. Abstract ______________________________________________________________________________ For decades transposon-based mutagenesis and transgenesis were used in invertebrate model organisms such as D. melanogaster and C. elegans. This approach offers numerous advantages over conventional methods of viral transduction or non-viral techniques. However, up until recently such methods were not available for vertebrates. Only after an extinct transposable element Sleeping Beauty (SB) was recovered from fish genome in 1997 situation has changed drastically. SB proved to be efficient in somatic, germinal and embryonic stem cells of different vertebrate model animals. Sleeping Beauty was found to be an indispensable tool for loss-of-function and cancer screens, generation of virus-free induced pluripotent stem cells (iPSCs) and human gene therapy. Further development of SB and other transposon systems in vertebrates will help to fill up the gap existing in
In relation to human cells
In relation to human cells:- Outline the microscopic structure and function of the main types of cells found in human bone. Bone cells are a very important feature of bones as they built tissue for support as well as actually forming and replacing bone itself. (Gunn, 2007) There are three main different types of bone cell in bone tissue. Osteoblasts, osteocytes and osteoclasts. (Thibodeau, 2007). All three of these cells differentiate in structure. Osteoblasts have a reticulum which is not very well developed. They have loose inorganic ctystals which clearing show bone destruction. Their structure like so, is organised for the synthesis of protein to form a bone matrix. (Cameron, Paschall and Robinson, 1967). They occur along a surface and usually lie adjacent to each other upon lamellae which they secrete. They are known as bone-forming cells and are small cuboidal cells in shape. (King., 2009). Osteocytes are small cells. They are secreted from osteoblasts are isolated from one another in individual lacunae. The cell has tiny canaliculi which extend out to undertake the cell processes. They are divided by gaps but are close enough for means of communication and nutrient supply with one another. (King, 2009). Osteoclasts can be known as the bone-removing cell. They are a large cell and have multiple nuclei. They occur in small hollows which erode into the surface.
The Effect Of Osmosis In Animal Cells, Plant Cells & A Model System
The Effect Of Osmosis In Animal Cells, Plant Cells & A Model System Introduction The purpose of this experiment is to determine as well as observe the movement of water molecules going in and coming out of model system, animal cell and plant cell through a process called osmosis. Osmosis is referred to the movement of water molecules through a semi-permeable cell membrane from an area of low solute concentration to an area of high solute concentration within a biological cell system (Karp, 2010). In other terms osmosis can also be referred to as the movement of water molecules through a cell membrane from an area of high water concentration to an area of low water concentration (Karp, 2010). In this lab the membrane of the cells that are tested are permeable to water molecules, but impermeable to solutes. Permeability refers to substances that can enter, while impermeability means that substances can not enter (Karp, 2010). Within this experiment when the process of osmosis is observed between two solutions, the solute concentrations between the two solutions are compared in either three ways; they can either be isotonic, hypertonic or hypotonic. An isotonic solution is a solution that has the same concentration of solute to the solution being compared with (Bowen, 2010). In this case, the concentration of solute in the cell is equal to the concentration of the fluid that
Using Spectroscopy to Evaluate the Absorption of Light for Different Substances
Using Spectroscopy to Evaluate the Absorption of Light for Different Substances Erin Laura and Polina, Section 13 BIOL 130, Monday 7:00-9:50pm Rm B2 151 Performed on October 20th, 2008 Introduction The purpose of experiment one was to determine the concentration of the unknown solution by using spectroscopy and comparing that substance to substances of known concentrations. In experiment two, the purpose was to discover the regions of the visible light spectrum are absorbed by both samples of chlorophyll. The spectrophotometer was an exceptionally useful tool for this lab. Spectroscopy is valuable for identifying substances through absorption of light which is done by measuring substances and comparing them to other known substances. Specifically a spectrophotometer is a device that measures the absorption of radiation at a particular wavelength. This is done by a light bulb shining, refracting its light into one beam, which then passes through an exit slit, then through the test solution and to a detector. This detects the amount of light that passed through the substance and a readout shows the amount of light that was absorbed (Jones, A, et al, 2007). Beer's Law states that concentration of a substance is directly proportional to its amount of light absorption (Department of Biology, 2008). The number of molecules of the solute is related exponentially to the amount
MELTING POINT OF ORGANIC COMPOUNDS
NAME : THAMARAI A/P RAJENDRAN ID NUMBER : 09ALB07214 LABORATORY 1A : ATOMIC STRUCTURE, BONDING AND PERIODICITY COURSE : BIOTECHNOLOGY (YEAR 1 SEM 1) EXPERIMENT 7: MELTING POINT OF ORGANIC COMPOUNDS. TITLE: Melting Point of Organic Compounds. OBJECTIVES OF EXPERIMENT: The objective of this experiment is to identify unknown organic compound by melting point depression method. Practice use of melting point apparatus by measuring melting point of some pure organic compound. THEORY AND BACKGROUND: The melting point of organic solid can be determined by introducing a tiny amount of the substance into a small capillary tube, attaching this to the stem of a thermometer centerd in heating bath, heating the bath slowly, and observing the temperatures at which melting begins and is complete. Pure samples usually have sharp melting points, for example 149.5°C-150°C or 189°C-190°C; impure samples of the same compounds melt at lower temperatures and over a wide range, for example; 145°C-148°C or 187°C-189°C. The contaminant that depresses the melting point and extends the melting range may be an indefinitely characterized resinous material or it may be a trace of a second chemical entity of melting point either higher or lower than that of the major component. Under equilibrium conditions (no super cooling) the temperature at which a pure solid melts is identical
Describe the structure of keratin and collagen and show how their structures are suited to their biological functions. Discuss one disease/ metabolic disorder that can arise from a structural defect in either protein.
Describe the structure of keratin and collagen and show how their structures are suited to their biological functions. Discuss one disease/ metabolic disorder that can arise from a structural defect in either protein. Keratin and collagen are very vital within the body's structure; both are very similar in nature, being that they derive from amino acids, which make up their respective proteins. They both fall under a group of particular proteins known as scleroproteins or fibrous in nature. Majority of their structure consist of left-handed helix structures1. Scleroproteins make up one of the two major protein categories, the other being globular. Keratin and collagen each represent one of the three subclasses within scleroproteins. As scleroproteins their jobs are to provide support and protection for the body. They are adapt for this because of their complex structure of repeating polypeptide chains which strengthen as the bind together; this and their insolubility are vital in maintaining the anatomy of the body. Whilst being similar in some ways, collagen and keratin remain different in their specific functions and role. As mentioned above keratin is one of the three scleroproteins. It is an extremely strong, insoluble tertiary protein; it is the main constituent in the structures of various parts in the body. These include the skin, hair, nails, as well as horns and