Protein Purification Methods And Structure Functions Biology Essay

Protein Purification Methods And Structure Functions Biology Essay Protein purification is the method by which particular protein of interest is being separated from a complex mixture. This is a very important process in order to understand the structure, function and interactions of particular protein. First of all separation of protein from the matrix containing it and then its separation from non protein parts and finally separating desired protein from other proteins. There are three general analytical purification methods on the basis of protein properties. Firstly on the basis of isoelectric point, then size or molecular mass and lastly on the basis of hydrophobicity or polarity. Purification of protein is generally dependent upon its properties which are determined by its amino acid sequence. (http://en.wikipedia.org/wiki/Protein_purification). Molecular parameters of human TNF alpha such as molecular weight and isoelectric point was studied by using the postparam program of ExPASY web tool. The molecular weight was found to be 58675.5 and th eoretical pI was 5.06. Purification The recombinant human TNF alpha is synthesized by the transformed K. lactis and was secreted out into the nitrogen free minimal medium because of ÃŽÂ ±-MF signal sequence present in the expression vector pkLAC2. Purification of human TNF alpha was done in few steps procedure. The first step in the purification of particular protein is its recovery from the medium and in this case TNF alpha is released outside of the cell in extra cellular medium. This is done by the help of centrifugation at 5000 rpm for 20 minutes to separate cells and the culture media. The desired protein is present in the supernatant from where it is collected and subjected to further purification. Affinity Chromatography The human TNF alpha protein has been tagged with the hemagglutinin (HA) peptide. In order to purify the protein, Pierce HA Tag IP/Co-IP Kit (Thermo Fisher Scientific, 2010) was used that provide affinity resin and other reagent to perform immunoprecipitation or co-immunoprecipitation reactions by using HA tagged protein as bait. This kit contains the prepared agarose affinity resin, buffers, microcentrifuge spin columns, a positive control and easy-to-follow instructions. Highly specific anti- HA anitibody is covalently immobilized on the beaded agarose in the kit. There is precipitation of HA tagged protein on the agarose after incubation. This specific interaction complex is being easily eluted from the resis by using simple washing steps and then is supplied with SDS-PAGE sample loading buffer for further analysis. SDS-PAGE After isolating and extraction of the desired protein, its purity is checked by using a technique known as SDS-PAGE (Sodium dodycyl sulphate polyacrylamide gel). SDS-PAGE is a technique used for the characterization and separation of peptides and proteins in a complex mixture. The characterization of SDS-PAGE depends upon the molecular weight of a particular protein and its electrophoretic mobility. The structure of protein also effects its migration of protein through the gel. Sodium dodycyl sulphate (SDS) is anionic surfactant produce a negative charge coat on the proteins. These charged particles move towards their opposite charge electrode under the influence of electric fluid during electrophoresis. The electrophoresis of the protein through the gel is majorly affected by the ionic composition, pH and temperature of electrophoretic medium. In electrophretic migration, the pour size of the gel is also one of the major factors that affect electrophoresis. (Creighton, 1998) After loading the sample, the gel was run at constant current for 2-3 hours. Uniform bands were observed on the gel indicating the purity of protein. Mass spectrometry Mass spectrometry is a technique which can be used to determine the purity of protein. As molecular weight of a protein is an important characteristic, that can indicate its purity. Mass spectrometer is a technique that detects mass to charge ratio of ions. The procedure involved is first loading of the sample for ionization in mass spectrometer. The molecules can be manipulated easily when they are ionized. These ionized molecules are separated on the basis of their mass to charge ratio in analyzer region. Afterwards, these separated ions are detected analyzed by the detector. Mass spectrometer works in a high vacuum, so that the traveling of the ions is not hindered by the air molecule during their passage from one end to the other. The mass obtain in the result is analyzed and compared with the known mass of TNF-alpha. In case of similarity in the masses, it is confirmed that the protein is completely pure. Any dissimilarity indicates that the protein is not completely purified. (Creighton, 1998) Biological Activity of TNF-alpha The biological activity of TNF-alpha can be determined by Human TNF-alpha Elisa kit (Signosis, Inc 2010). The principle of this assay is based upon solid phase ELISA. The mouse anti human TNF-alpha antibody is used in this assay for immobilization of TNF-alpha protein. For detection purpose, goat anti human TNF-alpha antibodies, which are conjugated to horseradish peroxidase (HRP) with streptavidin are used. The two antibodies and the sample are incubated for allowing the reaction to take place. It leads to a sandwich formation of TNF-alpha protein in between the two antibodies. The wells are then washed after incubation, so that the unbound enzyme-linked antibodies are removed. Tetramethylbenzidin (TMB) is added in the well which is a HRP substrate. It develops a blue color while reacting with streptravidin-HRP conjugate. A stop solution is then added which changes the blue color into yellow. It is then analyzed spectrophotometrically. Absorbance is measured at 450 nm. (Signosis, In c 2010) Component of Human TNF-alpha Elisa Kit Mouse anti-human TNF-alpha antibody coated 96 well microplate. Goat anti-human TNF-alpha antibodies, biotin labeled. Streptravidin-HRP conjugate. Substrate. Standard recombinant TNF-alpha (290ng/ml) Assay wash buffer. Blocking buffer. Stop solution.

Essay Invisible Hand Essay

The invisible hand is a metaphor coined by the economist Adam Smith. Once in â€Å"The Wealth of Nations† and other writings, Smith demonstrated that, in a free market, an individual pursuing his own self-interest tends to also promote the good of his community as a whole through a principle that he called â€Å"the invisible hand†. He argued that each individual maximizing revenue for himself maximizes the total revenue of society as a whole, as this is identical with the sum total of individual revenues. Smith used the term ‘invisible hand’ only three times, but the metaphor later gained widespread use Several new interpretations of Adam Smith’s invisible hand have recently been published in leading general-interest economic journals. These interpretations attempt to bring Smith forward in time, to make him more modern, and to fashion him in the image of the modern welfare theorist. Here we go back in time and find the source for both of Smith’s economic applications of the invisible hand in Richard Cantillon’s model of the isolated estate. With this connection established, we know what Smith read and dubbed the invisible hand. Introduction We now know a great deal about the intricacies and details of Adam Smith’s life and economics. Scholars have, for example, poured over his views on the organization of religion, his views of the corporation, and even his tenure as a tax collector, and have established definite conclusions. In contrast, Smith’s most famous concept—â€Å"the invisible hand†Ã¢â‚¬â€has in recent years been placed in an intellectual quagmire as a result of a surprising resurgence of interest in the meaning of the concept. Several new interpretations of the concept have been published in the leading general-interest economic journals, as well as those that specialize in the history of economic thought. This widespread effort to discover the â€Å"true† meaning of the invisible hand appears to have muddied the conceptual waters almost beyond recognition. There are now at least a dozen different versions of the invisible hand ranging from the more traditional interpretations to those which attach the phrase to such things as slavery and national defense. Smith’s invisible hand now suffers from multiple-conception disorder and the lack of an acceptable definition could render Smith’s concept scientifically useless. The opening quote from Khalil represents one of the few sensible modern interpretations of Smith (the process theorist) because it shows both how far modern interpreters have gone astray—painting Smith forward in time as a modern neoclassical welfare (end state) theorist, and why there is so much confusion—Smith’s three different uses of the phrase. To resolve the mystery of the meaning of the invisible hand, I would like to go backward in time and show that Smith discovered the general conceptual framework for the invisible hand in Richard Cantillon’s Essai sur la Nature du Commerce en General (hereafter, Essai). Cantillon’s model of the isolated estate represents a revolutionary breakthrough in economic theory and both of Smith’s economic applications of the invisible hand—which hitherto have been understood to be disconnected—can be found in it. This linkage between Smith and Cantillon permits us to describe the invisible hand as the processes that constitute price theory, competition, and distribution. First, however i will briefly describe the heated debate in the general-interest journals over the meaning of the invisible hand and then present the broader connections that scholars have made between Cantillon and Smith. Part I Understood as a metaphor Smith uses the metaphor in the context of an argument against protectionism and government regulation of markets, but it is based on very broad principles developed by Bernard Mandeville, Bishop Butler, Lord Shaftesbury, and Francis Hutcheson. In general, the term â€Å"invisible hand† can apply to any individual action that has unplanned, unintended consequences, particularly those which arise from actions not orchestrated by a central command and which have an observable, patterned effect on the community. Bernard Mandeville argued that private vices are actually public benefits. In â€Å"The Fable of the Bees† (1714), he laments that the â€Å"bees of social virtue are buzzing in Man’s bonnet†: that civilized man has stigmatized his private appetites and the result is the retardation of the common good. Bishop Butler argued that pursuing the public good was the best way of advancing one’s own good since the two were necessarily identical. Lord Shaftesbury turned the convergence of public and private good around, claiming that acting in accordance with one’s self-interest will produce socially beneficial results. An underlying unifying force that Shaftesbury called the â€Å"Will of Nature† maintains equilibrium, congruency, and harmony. This force, if it is to operate freely, requires the individual pursuit of rational self-interest, and the preservation and advancement of the self. Francis Hutcheson also accepted this convergence between public and private interest, but he attributed the mechanism, not to rational self-interest, but to personal intuition, which he called a â€Å"moral sense. † Smith developed his own version of this general principle in which six psychological motives combine in each individual to produce the common good. In â€Å"The Theory of Moral Sentiments†, vol. II, page 316, he says, â€Å"â€Å"By acting according to the dictates of our moral faculties, we necessarily pursue the most effective means for promoting the happiness of mankind. †Ã¢â‚¬  Contrary to common misconceptions, Smith did not assert that all self-interested labour necessarily benefits society, or that all public goods are produced through self-interested labour. His proposal is merely that in a free market, people â€Å"usually† tend to produce goods desired by their neighbours. The tragedy of the commons is an example where self-interest tends to bring an unwanted result. Moreover, a free market arguably provides numerous opportunities for maximizing one’s own profit at the expense (rather than for the benefit) of others. The tobacco industry is often cited as an example of this: the sale of cigarettes and other tobacco products certainly brings a very good revenue, but the industry’s critics deny that the social benefits (the pleasures associated with smoking, the camaraderie, the feeling of doing something â€Å"cool†) can possibly outbalance the social costs. Part II Economists’ Interpretation of The Wealth of Nations quote The concept of the Invisible Hand is nearly always generalized beyond Smith’s original discussion of domestic versus foreign trade. Smith himself participated in such generalization, as is already evident in his allusion to â€Å"many other cases†, quoted above. Notice that the invisible hand is here considered a natural inclination, not yet a social mechanism as it was later classified by Leon Walras and Vilfredo Pareto. Many economists claim that the theory of the Invisible Hand states that if each consumer is allowed to choose freely what to buy and each producer is allowed to choose freely what to sell and how to produce it, the market will settle on a product distribution and prices that are beneficial to the all individual members of a community, and hence to the community as a whole. The reason for this is that greed will drive actors to beneficial behavior. Efficient methods of production will be adopted in order to maximize profits. Low prices will be charged in order to undercut competitors. Investors will invest in those industries that are most urgently needed to maximize returns, and withdraw capital from those that are less efficient in creating value. Students will be guided to prepare for the most needed (and therefore most remunerative) careers. And all these effects will take place dynamically and automatically. It also works as a balancing mechanism. For example, the inhabitants of a poor country will be willing to work very cheaply. Entrepreneurs can make great profits by building factories in poor countries. But since they increase the demand for labor, they will increase its price. And since the new producers will also become consumers, local businesses will have to hire more people in order to provide for them the things that they want to consume. As this process continues, the labor prices will eventually rise to the point at which there is no advantage for the foreign countries doing business in the formerly poor country. Overall, this mechanism will cause the local economy to function on its own. In The Wealth of Nations Smith provides a metaphor that illustrates the simplicity of the principle: It is not from the benevolence of the butcher, the brewer or the baker that we expect our dinner, but from their regard to their own interest. We address ourselves not to their humanity but to their self-love, and never talk to them of our necessities but of their advantages. Nobody but a beggar chooses to depend chiefly upon the benevolence of their fellow-citizens. Part III Examples and arguments A very simple real world example of how the invisible hand is supposed to ork is the queue for a supermarket checkout. Each customer getting in line selfishly chooses to maximize his own interest, that is to checkout in the shortest time, regardless of the other customers. Their utility maximizing choice is to get in queue in the shortest line, this means that eventually customers queue up in lines all of the same length. Therefore even without the slightest direction and by following only their selfishness, the lines are all of the same length, which is clearly the most efficient disposition. (This examples also illustrates the ties between economics and game theory. Note that to reap these benefits, the market should at least exist — in the total absence of regulation, if people were allowed to cut the queue, the result of selfish pursuit of interests would be a crowded mess. Also, as this example also illustrates, economists have a particular understanding of efficiency. If a woman in the supermarket seeking to checkout is pregnant, carrying a crying child who is diabetic and who needs to eat dinner in the shortest amount of time possible, then it may be more efficient to allow her to jump the queue. Since Smith’s time, the principle of the invisible hand has been further incorporated into economic theory. Leon Walras developed a four equation general equilibrium model which concludes that individual self-interest operating in a competitive market place produces the unique conditions under which a society’s total utility is maximized. Vilfredo Pareto used an edgeworth box contact line to illustrate a similar social optimality. Ludwig von Mises, in Human Action, claims that Smith believed that the invisible hand was that of God. He did not mean this as a criticism, since he held that secular reasoning leads to similar conclusions. The invisible hand is traditionally understood as a concept in economics, but Robert Nozick argues in Anarchy, State and Utopia that substantively the same concept exists in a number of other areas of academic discourse under different names, notably Darwinian natural selection. In turn, Daniel Dennett has argued in Darwin’s Dangerous Idea that this represents a â€Å"universal acid† which may be applied to a number of seemingly disparate areas of philosophical enquiry (consciousness and free will in particular).

How is sociology important in your family Essay

John J. Macionis explains the importance of sociology in family in the thirteenth chapter, ‘Family and Religion’ of his book ‘Society, The Basics’. When the analysis is done on micro-level we learn that individuals get formed up and learn a lot through the experience of family life. Members of the family especially children build emotional bonds, become intimate with one another and above all are taught about their culture and values. Family gives an ideal picture of marriage and it is seen that matured individuals in United States find great pleasure in upbringing their own children. The structural functional approach of importance of family for society at macro level according to Macionis is, The family performs vital tasks, including socializing the young and providing emotional and functional support for members. The family helps regulate sexual activity. The social conflict approach is, The family perpetuates social inequality by handing down wealth from one generation to the next. The family supports patriarchy as well as racial and ethnic inequality. The symbolic interaction approach is, The reality of family life is constructed by members in their interaction. Courtship typically brings together people who offer the same level of advantages. (Society, The Basics, Chapter Thirteen, Family and Religion, Page- 24) Macionis explains how is sociology important to your family and family is also equally important to the society. In fact, describing the basic concepts of the family, Macionis explains that Family- a social institution found in all societies that unites people in cooperative groups to oversee the bearing and raising of children. (Society, The Basics, Chapter Thirteen, Family and Religion, Page- 2) As there have been development and changes in the society, family structure as well as cultural norms have also changed and modified. There have been both pros and cons of the development and they need to be understood in order to prevent the dark effects of progress on society and families as well. The family performs a lot of duties, which includes mainly of providing material and emotional security to its members and socialization. Due to increase in divorce rates and broken families these duties are not completely performed. Society depends upon families in many aspects and family on the other hand plays a role in social classification based on values and ethics. Macionis writes that every marriage consists of two dissimilar relationships in which one is that of men and the other of women. The expectations and anticipations of both the genders are different but marriage stays on the pillars of mutual understanding. People’s thinking have changed regarding having babies through new technologies of reproduction which help reduce the rate of birth defects and also help those couples have their own children who suffer with some deficiency or so. There has been stress on the importance of religion on society and individuals and history depicts that there have been many social reform movements that have been promoted due to religiosity. It is believed that outbreak of industrial revolution was because of religious principles and doctrine of Calvinism. There have been lots of problems and transitions in family life because history portrays women as property of men and domestic violence as a private concern and these concepts are changing with time. These are the points of discussion and detailed information found in this chapter, ‘Family and Religion’ from the book, ‘Society, The Basics’ by Macionis that I appreciate and like the most.

How Evolution Explains Zebra Stripes

It turns out that zebras are not referees at the horse games as many children may think. In fact, the patterns of the black and white stripes on a zebra are an evolutionary adaptation that has benefits for the animals. Several different and  plausible hypotheses have been proposed for the reason behind the stripes ever since Charles Darwin first came on the scene. Even he puzzled over the significance of the stripes. Over the years, different scientists have suggested the stripes could either be to help camouflage the zebras or confuse predators. Other ideas were to lower body temperature, repel insects, or to help them socialize with one another. The Evolutionary Advantage of Stripes A study, done by Tim Caro and his team from the University of California, Davis, pitted all of these hypotheses against each other and studied the statistics and data gathered. Remarkably, the statistical analysis showed over and over again that the most likely explanation for the stripes was to keep flies from biting the zebras. Although the statistical research is sound, many scientists are careful about declaring that hypothesis the winner until more specific research can be done. So why would stripes be able to keep the flies from biting the zebras? The pattern of the stripes seems to be a deterrent to the flies possibly due to the make up of the flies eyes. Flies have a set of compound eyes, just like humans do, but the way they see out of them is much different. Most species of flies can detect motion, shapes, and even color. However, they do not use cones and rods in their eyes. Instead, they evolved small individual visual receptors called  ommatidia. Each compound eye of the fly has thousands of these ommatidia that create a very broad field of vision for the fly. Another difference between human and fly eyes is that our eyes are attached to muscles that can move our eyes. That allows us to be able to focus as we look around. A flys eye is stationary and cannot move. Instead, each ommatidium collects and processes information from different directions. This means the fly is seeing in several different directions at once and its brain is processing all of this information at the same time. The striped pattern of a zebras coat is a sort of optical illusion to the flys eye because of its inability to focus and see the pattern. It is hypothesized that the fly either misinterprets the stripes as different individuals, or it is a sort of depth perception issue where the flies simply just miss the zebra as they try to feast on it. With the new information from the team at the University of California, Davis, it may be possible for other researchers in the field to experiment and get more information about this very advantageous adaptation for zebras and why it works to keep the flies at bay. As stated above, however, many scientists in the field are hesitant to back this research. There are many other hypotheses as to why zebras have stripes, and there may be several contributing factors as to why zebras have stripes. Just like several human traits are controlled by multiple genes, zebra stripes may be the equivalent for the zebra species. There may just be more than one reason as to why the zebras evolved stripes and not having flies biting them may just be one of them (or a pleasant side effect of the real reason).