Report Assignment Example | Topics and Well Written Essays - 3000 words - 1

Report - Assignment Example Any children’s right forums should relate existing human rights law to the particular circumstances of these children and develop existing laws to meet the specific needs of vulnerable children. It is the responsibility and rights of parents and educators to offer guidance in the implementation of rights of these children. They must develop an approach that, takes into account the child’s evolving capacities, such as age and self-realization. Self-identity can be explained as the descriptive characteristics, abilities, qualities, of a person. (Freeman, 2000) Listening to children talk about their right and rights of other, there is need to put in place more active ways of particularly identifying children’s views, mainly when it comes to conducting a research and educational practices. Children, and in particular young ones, should be allowed to express their views in any way possible even through children’s activities like drawing and orally. The meanings young ones attach to their experiences are rarely the meanings that the adults in charge of them would ascribe. (Bandman, 1999) The session will focus on the importance of understanding how children construct and develop their own sense of what their rights are and in what way they develop a sense of belonging within the community and family they come from. It will scrutinize the importance of early experiences in relation to their self-worth. The connection among the vision, belonging, and the shaping of children’s identity will be discussed, including the areas of attachment, social- cultural heritage, developing experiences and progressive relationship structure will be explored. Johnny is a seven-year-old boy in a kindergarten school, he is from a humble background, as the first-born child of four boys, and one girl he is curious about his surroundings. At this age, he can ask questions and seek answers about

Implied and Express terms (contract) Essay Example | Topics and Well Written Essays - 1000 words

Implied and Express terms (contract) - Essay Example In such situations the concept of what are implied terms comes into the picture. Implied terms could be a condition, a warranty or an innominate term and it’s on only by knowing the effect of each, that we could determine the distinction of one from the other. The court, in the case of Hong Kong Fir v Kawasaki Kisen Kaisha [1962] 2 QB 26, had the chance to make a distinction among a condition, warranty and an innominate term. In said case, the following definitions were settled: When a condition is breached the injured party has the right to sue for damages and also to terminate the contract. A breach of warranty only gives rise to the right to sue for damages. When an innominate term is breached the legal consequences of the breach depend upon its factual consequences i.e. there is a right to terminate the contract, in addition to suing for damages, only if the breach of an innominate term is such as to deprive the injured party of substantially all the benefit which he was intended to derive from the contract. If a term is subject to different interpretation then there is flexibility otherwise there is certainty. As to how it creates a tension is on the consequence of different interpretations. A party to a contract, for example, believes a term to be condition but when the court will interpret otherwise, it will really create a tension as to him because that would be depriving him the right to terminate the contract. a or exanmplke iif one entitles meFF Since in innominate terms there could be either the right to sue for damages or both the said right and right to terminate the contract, then a tension could also be created by the uncertainty of flexibility of the decision depending on how the courts appreciates the facts of the cases and surrounding circumstances and how it will apply the rule on implied terms. As to why, the court said: ‘Terms implied in fact are

Managing people

Managing people Introduction: Leadership is integrated part of our life. According to corporate chief and former US presidential candidate Ross Perot, â€Å"the principles of leadership are timeless because, in a rapidly changing world, human nature remains a constant†. We all experience leadership in our life from early childhood in our families, through friendships, social, recreational and sports activities, school and higher education, to politics and government, and, of course, in our work, we all recognize leadership in other people and often in ourselves. In government, global corporations and small businesses alike, the leadership role is becoming more demanding, more open to scrutiny and more difficult [Roger Gill]. The development of leadership theory also parallels the development of organizational theory. The bureaucratic form of organization is characterized by ‘laissez-faire leadership whereby so-called leaders tend to avoid taking a stand, ignore problems, not follow up, and refrain from intervening or transactional leadership, in which leaders practise management by exception, focusing only on deviations from what is required, and contingent reward, rewarding people (either materially or psychologically) for achieving what is required. The emergence of the post-bureaucratic form of organization in the late nineteenth century reflects the development of the concept of transformational leadership. Theory Approaches to Leadership:Number of Leadership theories and approaches has been evolved on the basis of Style, Trait, Behavioural, Transformational, Situational and Charisma. Many researchers made efforts linking some of the theories across these leadership approaches. But each model has its own pros, cons, assumptions and limitations. Latest researches are conducted on Situational Transformational leadership styles. Leadership gurus presented new models as variations to the already existing models. Max Weber, MacGregor, Bernard Bass, Warren Bennis Nanus are few important researchers in the area of transformational leadership. Understanding the difference between transactional and transformational leadership is vital in getting the whole concept of transformational leadership theory. In general, a relationship between two people is based on the level of exchange they have. Exchange need not be money or material; it can be anything. The more exchange they have the more stronger the relation. Managers expects more productivity from employee in order to give good rewards. In this way, if something is done to anyone based on the return then that relation is called as ‘Transactional type. In business, leaders announces rewards in turn to the productivity. These relations are all about requirements, conditions and rewards. In life, at one point of time, things happen without expectation from other side. Say, moms dedicated service to her kid. Mom doesnt expect anything from the child and the service she provides in raising the child isunconditional, dedicated, committed. Mom plays a major role in shaping up the kids future life. This type of relation is called as ‘Transformational. Leaders do exist in this world with these behaviours. Transformational Leade rs work toward a common goal with followers; put followers in front and develop them; take followers to next level; inspire followers to transcend their own self-interests in achieving superior results. Leadership Approach in TATA Group: TATA Group founded in 1868, is an Indian multinational conglomerate headquartered in the Mumbai, India. The Group has 500,000 employees spread over six continents (more than 80 countries). TATA Group has market capitalization worth $70bn as of today and is the largest private corporate group in India. TATA Group is biggest employer in UK, employing more than 50,000 people. TATA Group has interests in communications, IT, engineering, materials, services, energy, consumer products and chemicals. Its chairman, Ratan Tata is one of Indias and the worlds most influential person right now. The Tata Group is known for its good business ethics and corporate governance. The Group leadership style has been quite consistent from its existence. The Group has incorporated some more leadership changes which are essential in current century to drive towards more competitive. In terms of leadership style, TATA Group has adopted a team-led culture. With Ratan as a leader, the management style of the entire group changed, trust became a huge facet and theme of the group. Ratan put in a complete organisational restructuring in place when he took over taking a more matrix-style approach building teams, replacing many of the senior managers with younger ones and bringing the retirement age of senior managers to 65 from 70 years. These changes would have obviously transformed a lot in the business, senior managers would have had to be on their toes and flexibility and adaptability became essential qualities to have. The leadership changed from a centralised, command centre to a much more distributed form with employees and all managers enjoying greater responsibility and knowledge about the Group, which would have in turn; motivated them to work harder and as a group. From distinctive Leadership models available such as the McGregor Theory X and Y; where a theory X manager believes workers dislike work, are not creative and avoid al l responsibility while a theory Y manager believes that workers get as much enjoyment from work as they can derive with leisure, accept responsibility and are creative; it can be seen from this, that Ratan wanted all his managers to be modelled as closely to Theory Y and he himself could be called a Theory Y manager. He encouraged managers to be innovative and share all their ideas, consulting actively with them and giving them more responsibility and importantly encouraged team-working. Using standard leadership style models of Autocratic, Democratic, Paternalistic and Laissez-faire, Ratan Tata can be described as the leader who employs a more democratic approach but also uses facets of the other three models, a perfect leader in my view. He is democratic because he encourages communication and participation, and workers have access to some of the knowledge of the business. However, he is also paternalistic in a way, because he cares greatly about the well-being of his employees as was evident because after the 26/11 terror attacks, he personally visited each affected employees residence (80 in total) which shows he does have a humanistic touch to his leadership as well. He is autocratic in certain ways but only when needed especially when quick and informed decisions have to be taken, but he is never too commanding in his nature, being a man of few words and being more of a man of action, this is evident from the manner he aggressively pushes for bold international deals, such as during the global acquisitions of business powerhouses such as Corus, Jaguar and Land Rover, and Tetley Tea. This quote from Mr. Muthuram, another executive director, clearly shows that he is a man, who is intent on succeeding and is sure of himself, â€Å"Ratan was the chief architect of the Corus deal. I was worried about the magnitude and the amount of money. But he instilled confidence.† He also uses facets of the Laissez-Faire model such as the delegation of important duties and decision-making, he also does not in any way interfere with any managers functioning, he might make a broad strategic assessment but he does not interfere in operational issues and details, this shows that he has complete trust and faith in his managers and believes in their ability, this quote from Mr. Gopalakrishnan, an executive director of the company, shows how much value Ratan Tata places on his trust, this can be highly motivating for managers and workers alike, I remember what Mr Tata told us at a meeting. He said that he will continue to trust all his managers, but once they lose that trust, he will go after them. I think that is a very fair deal† Looking at other leadership models, such as Max Webers Transactional and Transformational Leadership models, where a leader is classed in three brackets which are Bureaucratic, Charismatic and Traditional, where a bureaucratic leader is one who is always bound by the set rule and does not want to tread beyond them; a Traditional leader is one who does and follows everything from a long past or history and always loyally obeys these ‘traditions; a Charismatic leader is one who uses his own laurels or abilities to inspire and is one who can be described as radically opposed to administrative rules and legal principles. From these models Ratan Tata easily falls into the Charismatic Model because he is one who leads by example, coming up with highly innovative ideas such as the one lakh car the ‘Nano, budget hotels or low-end watches, he brought radical change to the Tata Group as a whole, changing it from its ‘Traditional mindset to new more flexible and adaptive cultu ral mindset. One can also look at Bennis and Nanuss Transformational leadership model which states that transformational leaders make their followers into self-empowered leaders and their main focus is to articulate vision and values clearly so the newly self-empowered leaders know where to go; it then talks about the four Is of Transformational leadership which are Idealised Influence ( being a role model) Inspirational Motivation( cultivating a team spirit, motivate and provide a challenge) Intellectual Stimulation( Innovation and creativity) Individual Consideration(mentoring and providing support for followers) Ratan Tata can be then described as a complete transformational leader, because he epitomises all the Is and is clearly a man with a great vision; he changed the business culture to one that is team-based, he empowers all his managers and executives and has complete faith in them, he is extremely innovative and is credited for much of the Groups new products, he places a great deal of importance to his Research and Development department and he definitely cares deeply about the welfare of all his employees and managers, which was demonstrated during the 26/11 terror attacks that hit Mumbai and targeted one of his hotels. He is a visionary and proof of this comes from this quote, â€Å"One hundred years from now, I expect Tatas to be much bigger, of course, than it is now. More importantly, I hope the group comes to be regarded as being the best in India. Best in the Manner in which we operate, Best in the products we deliver, and best in our value system and ethics. Having said that, I hope that a hundred years from now we will spread our wings far beyond India, that we become a global group, operating in many countries, as Indian business conglomerate that is at home in the world, carrying the same set of trust as we do today† Ratan Tata is a leader who realised the kind of market his businesses were in competitively and always wanted them to be up to date in all their processes and technology. This famous quote from his lips is proof enough of this fact, A company or business which remains static is a business that will die; a company that constantly changes and accepts that there are better ways to do things than the way they are done today, is a company that will survive in the global market that we face. From this statement we can figure that he is a fierce competitor and a man who understands the market he faces. Ratan Tata. He is also a very deep thinker and a brilliant strategist as is described by one of his Executive directors, Mr. Alan Rosling, â€Å"He is a deep thinker and extremely strategic. He is always 2-3 steps ahead† Another important quality that Ratan Tata has is that he is a man of strong Integrity and principle; he never compromises on his ethics and does not deal with any business or company that compromises them and values his shareholders very highly , another quote from Mr. Gopalakrishnan shows this fact, Tata has shown that there is no other way he will do business other than do it ethically There is evidence of this fact from the incident when the finances of Tata Finance were in huge losses due to financial faults made by senior officials; Ratan announced that the holding company they owned would provide the necessary finance needed for the unascertained losses, thus giving shareholders their dividend. His personality is reflecting in the Groups reputation and giving them a good global image. I choose Ratan Tata because I feel he is a complete leader and he is someone who definitely has transformed the face of the Indian business world, he is someone who never compromises on his ethics and principles and has set a strong value system for the Tata Group as a whole, in a country where ethical and value-orientated business is never a top priority. He is someone who epitomises the complete leader with all his qualities; he is a man of great integrity and dignity, and he is also a champion of social causes with his the Tata Foundation being the largest charitable organisation base in the country, and he is a man of modesty who never likes to take credit for anything even if it does completely belong to him. Critically evaluate what is the relationship between Prediction Markets and the concept of Open Innovation. Answer to Question 2: There is a very strong fundamental relationship between Prediction Markets and the Concept Question 3: Discuss whether and to what extent business organisations can make use of Prediction Markets and critically evaluate what are the necessary modifications that organisations have to put in place in order to profit from the use of Prediction Markets. Answer to Question 3: In comparison with traditional methods to demand forecast, Prediction Markets provides

The Character of Casey in The Grapes of Wrath :: Grapes Wrath essays

The Character of Casey in The Grapes of Wrath John Steinbeck passionately describes a time of unfair poverty, unity, and the human spirit growth in the classic novel, The Grapes of Wrath. The novel tells of real, diverse characters that experience growth through turmoil and hardship. Jim Casy, a personal favorite character, is an ex-preacher that meets with a former worshiper, Tom Joad. Casy continues a relationship with Tom and the rest of the Joads as they embark on a journey to California with the hopes of prosperity. Casy represents how the many situations in life impact the ever-changing souls of human beings and the search within to discover one's true identity and beliefs. Casy, however, was much more complex than the average individual. His unprejudiced, unified, Christ-like existence twists and turns with every mental and extraneous disaccord. Jim Casy is an interesting, complicated man. He can be seen as a modern day Christ figure, except without the tending manifest belief in the Christian faith. The initials of his n ame, J.C., are the same as those of Jesus Christ. Just as Jesus was exalted by many for what he stood for and was supposed to be, Casy was hailed and respected by many for simply being a preacher. Casy and Jesus both saw a common goodness in the average man and saw every person as holy. Both Christ and Casy faced struggles between their ideals and the real world. Despite Casy's honesty, goodness, and loyalty to all men, he would not earn a meal or warm place to stay. Although Jesus had many followers, still others opposed his preaching until the very end.   These prophets attempted to disengage man from the cares of the world and create a high spiritualism that stemmed joy from misery. All the migrants found pleasures along their trips and kept their hope and spirit throughout the journey. Thanks to Jesus, the saddest, dullest existence has had its glimpse of Heaven. Casy once remarked, I gotta see them folks that's gone out on the road. I gotta feelin' I got to see them. They gon na need help no preachin' can give 'em. Hope of heaven when their lives ain't lived? Holy Sperit when their own sperit is downcast an' sad?" (page #)   Casy wished to reach out to others in spite of his own troubles. He wanted to give them sprit; hope and he wanted to rejuvenate their souls.

Psychology Essay

This required Portfolio assignment will provide you with the opportunity to practice and hone your research skills. It has been designed to help you think scientifically about real world problems and issues and to apply your knowledge of the research process to various topics in Psychology. This assignment accomplishes that goal by challenging you to: †¢ Differentiate between the common use of the word research and the use of the word research in the social and behavioral sciences †¢ Identify the major steps in the research process using a classic study in Psychology as an example. Part I: Defining Research The word research is used in many different ways. Consider the following examples: †¢ Your friend tells you that he intends to research different hair products before deciding on one to buy. †¢ A real estate agent advises you to research home values in your neighborhood before putting your house on the market. †¢ A police officer reports that she is doing ‘some research’ on possible motives for a crime that was committed. †¢ A writer states that he does ‘extensive research’ before beginning his fictional works. Answer the questions below: 1. How is research defined in the social and behavioral sciences? 2. What makes scientific research different from the examples provided above? In your response, be sure to address the characteristics of ‘good’ psychological research. Part II: Understanding the research process Researchers in Psychology follow a systematic process of investigation. Carefully read Chapter 2 of your textbook, paying special attention to the section on Experimental Research. Then go to Chapter 7 in your textbook and read the following section: Research In-Depth: Counterfactuals and â€Å"If Only†¦Ã¢â‚¬  Thinking. Answer the questions below, using Medvec & colleagues’ first study as an example: 1. What hypothesis did Medvec & colleagues set out to test in their first study of the ‘near miss’ phenomenon? Describe the theory associated with this hypothesis. 2. Identify the variables in the study and describe how they were measured. How did the researchers operationalize (test or measure) affective response upon winning a bronze or silver medal? 3. Who were the participants in the study and what did they do? 4. Describe the data that were collected and analyzed. 5. Describe the results of the study. What did the researchers conclude? 6. If you were to design a follow-up experiment on this subject, what might it be?

GFP protein

Green Florescent Protein, abbreviated as GFP, is a protein composed of 238 amino acids that is commonly found in mnemiopsis, comb Jelly. It has a major wavelength at 396 nm and a minor one at 475 nm. GFP is what gives mnemiopsis their bright green florescent glow. ultraviolet light, or blue light, is necessary to see the florescent glow of this protein. GFP is an irregular protein because It Is highly resistant to denaturation by temperature and PH. It can survive In temperatures up to 98 degrees and has a pH of 12. 2 due to Its complex exterior, called the beta barrel. At an pH higher than 12. It denatures. It also has an Isoelectric point at 5. 3. The peripheral beta barrel cannot be digested or broken apart by protease because of the strong bonds holding It together. The beta barrel protects the chromophore, which Is the substance which gives GFP Its green glow. When CFP Is extracted from the plasmid of an E. Coll or from a Jellyfish, It contains an array different contaminants ma king it difficult for scientist to do experiments with GFP. A procedure in purifying GFP from a crude cell extract to nearly 100% GFP so that it can be analyzed and used in scientific experiments and research is necessary.The goal is to ptimize each protocol used to purify crude GFP. Methods Ammonium Sulfate Precipitation To purify the crude samples of GFP, the ion exchange method separates substances inside the test tube by similar charge. A sample of crude GFP of 7. 5 mL in a plastic tube was used for the experiment. Knowing that 43. 6 grams of ammonium sulfate in a 100 mL solution yields a 70% percent saturated solution, the proportion 43. 6g 11 00 mL=x/7. 5 mL was used to determine that 3. 27 grams of ammonium sulfate needs to be added to the experimental sample. After adding the ammonium sulfate, the solution was stirred gently to prevent frothing.Once most of the solution is transferred, the tube was placed on a triple beam balance along with another tube that went through the same process. The centrifuge was set at 15,000 rpms for 15 minutes so that the hydrophobic materials will separate and become the supernatant while the GFP pellet will remain behind. Once the 15 minutes elapsed, a new pipette was used to remove the supernatant, leaving behind the pellet of GFP and hydrophilic contaminates. To remove the hydrophilic substances, 5 mL of 4 molar ammonium sulfate and 15 mL of 10 mL tris at a p of 8 was added Into the oak ridge entrifuge test tube.The solution Is then put Into the centrifuge at 15,000 rpm for 15 minutes again. Once 15 minutes has passed, the supernatant, containing the GFP, was removed by a pipette and put In a microfuge. Hydrophobic Interaction Hgure yaropnoDlc Interactlon set up One molar ammonium sulfate was added to the column to wash the sample. Adding 1 molar ammonium sulfate washes the sample because a high salt concentration increases hydrophobicity of the GFP and the buffer, causing most of the GFP to be at the very top of the column. Substances that are hydrophilic get flushed out of the olumn while the more hydrophobic substances stay in the container.After the column has been eluted with 1 molar ammonium sulfate, the tris buffer is added to the ammonium sulfate to dilute it into . 5 molar ammonium sulfate. The volume of 1 molar ammonium sulfate inside the oak ridge centrifuge test tube is the volume of the tris buffer that will be added. After the column chromatography has been flushed with . 5 molar ammonium sulfate, more hydrophobic substances will be flushed out since the hydrophobicity of the tris buffer and the GFP has decreased. This causes the GFP to spread out in the column. Finally the amount of . 5 molar ammonium sulfate is diluted with tris buffer to . 5 molar ammonium which should cause most of the GFP to be flushed out of the column along with other substances that are very hydrophobic. While this experiment is going on the liquid that comes out of the column is collected in multiple test tubes. These test tubes contain GFP and other contaminants. The solutions are than read by a spectrophotometer. Each test tube will be tested by the spectrophotometer so that a graph can be made. Anion Exchange Figure 2: Siphon Bridge set up for Anion Exchange Figure 3: Centricon Test Tube In order to use anion exchange, the starting condition of the sample needs to be in a low salt solution.However after the GFP had gone through hydrophobic interaction, it was in a high salt solution. Before facing this dilemma, the fractions were pooled by centricon which decreases the overall sample volume by removing some buffer and salt solution. This greatly increases the GFP concentration in the samples. The fractions are placed in the centricon and then into a centrifuge for 25 minutes at 3,000 rpm to be separated by size. The large proteins stay in the entricon while buffer and salt solution goes into the plunger. To reduce the concentration of salt in the GFP sample, the sample is diluted 10 folds.Since the amount of GFP that was restored was 18 mL, 162 mL of tris buffer needed to be added. The diluted GFP is then put in the chromatography container, containing positively charged DEAE which is attracted to the GFP at a low salt concentration. After the GFP has been completely filled, the column is connected to a beaker that contains a low salt concentration. the low salt concentration beaker is connected to a high salt concentration beaker. As one drop of low salt solution goes into the chromatography column, one drop of high salt solution goes into the low salt solution.Gradually the salt concentration increases in the low salt beaker and in the column chromatography, causing GFP to spread down the container. The eluted GFP dripped out of the column chromatography to be collected in test tubes. I nree pnase partltlonlng Figure 4: Precipitate of GFP. T-butanol is one top while contaminates are on bottom GFP then went through three-phase partitioning, also known as TT P. The fractions taken after an anion exchange was 15 millilieter. Ten ml of 4 M ammonium sulfate was added to this volume to increase the salt concentration of the solution to 1. M, which is about 40% salt saturation. Twenty-five milliliters of t-butanol was added then added which was the same amount of ammonium sulfate and GFP in the container. The container was then placed in the centrifuge for ten minutes at 4600 RPM, causing the mixture to split into three layers; butanol on top, GFP in solution on the bottom, and precipitated contaminants in-between. The top layer of butanol and disk of precipitate were taken out. The volume of GFP solution was again matched in utanol and the container went into the centrifuge again. An aspirator was used to extract the GFP into a microfuge. . 6M ammonium sulfate was then added to the microfuge and the container was placed in a micro centrifuge for one minute at 13,000 RPM. Butanol and other contaminants that had not been take out previously f ormed a disc, was then taken out with an aspirator and the remaining GFP was then left in the microfuge. HPLC Figure 5: HPLC basic layout After the sample went through three phase partitioning, it was put through the High Performance Liquid Chromatography for a final purification. First liquid was put into the HPLC to clean out any previous GFP inside the loop of the HPLC and the column of the HPLC.Then, GFP in the microfuge was sucked into an injector to be put into the HPLC. Pushing the top of the injector slowly, GFP entered into a loop inside the HPLC. Once the GFP was placed in the loop, a knob was turned clockwise to the word lock. The GFP was then sent to the column where it was purified further by size through the minuscule beads. About 6,000 pounds of pressure per square inch was produced by the HPLC to push the GFP through the beads. While this was occurring, a pectrophotometer connected to the HPLC read the wavelengths of substances.Near the 396 nm wavelength, GFP was col lected in a microfuge tube. A UV light was held near tne exlt 0T Results e HPLC to measure tne amount ng sample. Graph 1: Results of the sample after HIC at a wavelength of 395 nm Graph 2: Results of the sample after HIC at a wavelength of 280 nm Graph 3: Results of the sample after HIC of the entire spectrum Seventeen test tubes were received after the HIC purification process. A blank consisting of tris buffer and ammonium sulfate was sampled in the spectrophotometer against liquid from each of the seventeen test tubes.Graph one represents the sample after HIC at a wavelength of 395 nm while graph two Results shows the results after HIC at a wavelength of 280 nm. After HIC, the fractions 12 to 16 were chosen for their purity and recovery of GFP. Graph one show the amount of GFP in each fraction number while graph two shows the total amount of protein in each fraction number. Graph three shows the spectrum of the entire sample. Graph 4: Results after Anion Exchange at a 397 nm wave length Graph 5: Results after Anion Exchange at a 280 nm wavelength Graphing 6 Thirteen test tubes were collected from the Anion Exchange purification process.This time the samples were blanked against tris buffer at 8. 0 pH and 0. 5 molar sodium chloride. Graph four shows results of the Anion Exchange at a 397 nm wavelength and graph five shows the results after Anion Exchange at a 280 nm wavelength. Once again, the graph at a 297 nm wavelength shows the amount of GFP while the graph at a 280 nm wavelength shows the amount of total protein. Graph six represents the results of the entire spectrum. The GFP peak was a lot more visible. Step Iotal sample (mL Abs (280) Total Protein Abs (397) GFP Ratio Crude sample 120 1600 . 25 At-ns042- 20 1 . 61 . 9 118 HIC 18 . 28 . 173 . 618 15 . 126 . 130 1. 03 3 Phase Partitioning . 01 n/a . 75 . 243 . 257 1. 06 Table 1: This is the overall data table. The second column shows the total volume at the start of each purification step. The following two columns are the peaks of the graphs at those wavelengths. The last column represents the ratio of GFP to the total Protein. The most desirable ratio is 1. 25. Dlscusslon The first method in purifying the crude GFP was using the ammonium sulfate precipitate. When ammonium sulfate is placed in water, it dissociates into ammonium (NH4+) and sulfate ions (S042-).Water, composed of two hydrogen ions and one oxygen ion, is a polar molecule because the oxygen has a high electronegativity. Oxygen has a greater affinity making the oxygen portion of water negative and the hydrogen portion of the water positive. The dissociated positively charged ammonium ion is allured to the negatively charged oxygen while the negatively charged sulfate ions are attracted to the dissociated positively charged hydrogen. The attraction between the ammonium sulfate and the water was so strong that the GFP and other proteins were left unoccupied, causing them to precipitate.When GFP in the 70% salt solution was placed into the centrifuge, substances such as DNA and RNA was removed because they became part of the supernatant. At a 70% salt concentration, only hydrophilic substances stay in solution while the more hydrophobic substances precipitate. When the GFP in a 25% solution of salt was placed in the centrifuge, the GFP and other substances went back into solution because there not enough water was occupied by the salt. Before the GFP is placed in the centrifuge, it must be balanced with another centrifuge with the same weight and the two containers must be placed across from one another.This is vital because the centrifuge needs to be balanced when it is rotating at an incredibly fast speed. Failure to have balanced centrifuge containers can result in a broken centrifuge and loud sounds. Also when mixing the GFP with salt, it is important not the shake the container or frothing will occur, making it difficult to transfer the solution in to an oak ridge centrifuge tube. The second p urification procedure that GFP underwent was hydrophobic interactions. During this purification, GFP binded to the non-polar Phenyl Sepharose beads because of its non-polar and hydrophobic traits.However the water in tris buffer is strong enough to separate the attraction between GFP and the Phenyl Sepharose. Therefore a high salt concentration is necessary to occupy the water so that the GFP and the Phenyl Sepharose to be attracted together. At a high salt concentration, GFP with bind easily to the Phenyl Sepharose since very little water molecules would interfere with the attraction and at a low salt concentration, GFP would not bind easily to the Phenyl Sepharose because tnere wlll De a lot 0T unoccuplea water molecules tnat wlll De aDle to InterTere wltn the GFP and Phenyl Sepharose attraction.Before the experiment, ten millimolar tris buffer at a pH of 8 was used to clean the column in order to keep the pH stable and to wash away the salt, ammonium sulfate, in the column. Remov ing the salt is vital because the buffer that once surrounds the salt will be allured to the hydrophobic benzene and to the hydrophobic patches on the GFP. Since the hydrophobic patches of the GFP are already filled, they will be flushed out, leaving mostly beads of benzene and the 10 millimolar tris buffer at a pH of 8. Once the column has been clean, it needs to be equilibrated so that the salt concentration is the same through the olumn.The step gradient used, started ata 1 molar ammonium sulfate concentration and was halved until a . 25 molar concentration to separate substances by hydrophobicity. The third purification procedure was anion exchange. In this procedure, GFP and other contaminants are separated by charge. The beads in the containers are different from the beads from the hydrophobic interaction because on they have a different chemical called DEAE which makes them positively charged. GFP has both protons and electrons on it which is why it was not easily attracted t o the DEAE, which is why the GFP is put in a basic solution.Ata high pH, the amount of negatively charged hydroxide increases and these hydroxides are allured by the protons on the GFP. The protons are than neutralized, making GFP a negatively charged molecule. The isoelectric point of GFP is at a pH of 5. 3. Ata pH higher than 5. 3, it is negatively charged and when it is at a pH lower than 5. 3, it is positively charged. Once the column chromatography is filled with GFP and connected to a beaker of low salt which connected to a beaker of high salt, anion exchange occurs. As the salt concentration increases, the GFP slowly spreads down the column and eventually out f the column into test tubes.Between the HIC and the Ion exchange chromatography, the sample the fractions were pooled and put in a centricon causing the GFP concentration in the samples to increase. This occurred because the ultrafilter only allowed particles smaller than protein to go in to the pusher. The large protei ns stay in the centricon while buffer and salt solution goes into the plunger. The sample of GFP was also diluted 10 folds because the sample needs to be in a low salt solution to use anion exchange and after the GFP had gone through hydrophobic interaction, it was in a high salt solution.The anion exchange method creates a continuous salt gradient because as one drop of low salt solution goes into the column chromatography, causing GFP to spread down the container. The follow procedure was the three phase partitioning purification. T-butanol and 1. 6 molar ammonium sulfate were essential for this procedure. T-butanol has a low density causing in to stay above the GFP solution. In addition it has an attraction for water and other hydrophobic substances causing 5 mL of water to be drawn out of the GFP sample and precipitated substances to float between the t-butanol and the GFP sample.Fresh t-butanol is necessary after removing the old t-butanol with the contaminants because at that point, the salt concentration had increased since water was drawn out. was aDle to De preclpltatea Decause 0T tne nlgn salt concentration. The final procedure for purifying GFP was using the HPLC which separated substances by size. The beads used in the HPLC column are miniscule and porous. The pours on the beads give substances of the same size more opportunities to leave the HPLC at the same time. Since the beads are so small, high pressure is needed to push the GFP sample through the beads.Naturally, smaller substances will exit the HPLC first while larger materials will exist last. In all scientific experiments room for error is unavoidable. During the HIC, IEX, three phase partitioning, and the HPLC, amounts of GFP were lost due to the GFP sticking to a container, a pipette, and even spills. During the HIC some of the GFP was lost due the overflowing the test tubes with liquid exiting the column. During the HPLC some GFP was lost because not all GFP dripping out of the HPLC wen t in to the microphage. Other errors include letting the column dry because the liquid was not dded to the beaker about the column.During the spectrophotometer runs, the blank was no inserted correctly causing the reading of the GFP to be incorrect. In addition, the order in which the GFP samples were suppose to be placed in the spectrophotometer was messed up. Judging from the overall purification table, table 1, the purification was quite successful. Originally, the ratio was only . 25, but by the end of all the purification procedures, it obtained a ratio 1. 06. A 1. 25 ratio is most desirable and through the purification, the ratio was nearly reached. The anion exchange, three phase artitioning, and the HPLC purification were the most impacting procedures.The anion exchange greatly increased the purity of the crude sample compared to the HIC purification. The three phase partitioning and HPLC purified the GFP even more. Some improvements to the protocols would be to start with t he anion exchange purification so that overall, the salt solution would go from a low salt concentration to a higher salt concentration. This also eliminates the need to dilute the solution. In addition, an automatic machine could be used to shift the test tubes that collect the iquid exiting the columns to prevent overflowing test tubes and the risk losing GFP.GFP is unique because of its florescent glow. This glow can be used as a marker or an indicator. If a glowing marker could be placed on infectious cells such as tumor cells or cancerous cells, it would revolutionize the treatment of these diseases because doctors will be able to track where the harmful cells are. In addition, if it is possible to trigger the florescence of GFP with UV light, it can eventually be used in light bulbs to produce light. GFP light bulbs would last for an incredibly long time ince they are very resistant to denaturing.In addition, in vehicles, GFP can be mixed in the motor oil, transmission oil, po wer steering oil, air conditioning oil, and other oils so that if a leak occurs in a car, it can easily be spotted by shinning UV light on the car. The purification of GFP can lead to endless new innovations in electrical engineering, automotive repair, and curing deadly diseases.