Isolation of Protein by Ammonium Sulphate Precipitation

Isolation of Protein by Ammonium Sulphate Precipitation Hypothesis Rubisco is a negatively charged protein that weighs 55,000 kDa and is also very soluble. When we add ammonium sulfate to reach a saturation of 50%, Rubisco can be isolated using ion exchange chromatography and protein electrophoresis. Materials and Methods Isolation of Protein by Ammonium Sulfate Precipitation Approximately 300g of fresh spinach leaves were de-ribbed and dried, then homogenized for 1 minute in 200mL of buffer. From the homogenized solution, approximately 50ml was placed into a beaker, which was stirred on a stir plate while 10.90g of solid ammonium sulfate was slowly added to reach 37% saturation. The solution was stirred for an additional 10 minutes and then centrifuged at 9,000-xg for 15 minutes. The pellet (Pellet I) was resuspended in 4mL of water and transferred into a dialysis bag for dialysis against distilled water. The supernatant was poured into a beaker and stirred on a stir plate while about 3g of ammonium sulfate was slowly added to reach 50% saturation. After about 15 minutes of stirring, the supernatant was centrifuged for 15 minutes at 7,000-xg. Following centrifugation, the pellet (Pellet II) was resuspended in 4mL of water and transferred into a dialysis bag for dialysis. Ion Exchange Column Chromatography The column was equilibrated by running 30mL of Buffer A (10mM Tris pH 8.0, 3mM EDTA) through. Afterwards, a centrifuge was used to pellet down any solid precipitate out of the dialyzed samples. 1mL of each sample (Pellet I and II) was transferred into labeled Eppendorf tubes and frozen for later use in the SDS-PAGE. Pellet I was diluted a 100-fold and about 4mL of diluted Pellet I and undiluted Pellet II were loaded into separate columns and the samples were allowed to flow through. The flow-through was discarded. 10mL of the low salt buffer (Buffer A + 50mM NaCl) was loaded onto the column and fractions of approximately 2mL were collected in separate cuvettes and labeled in order. After blanking the spectrophotometer at 280nm with low salt buffer, the OD readings of each fraction was measured. This process was repeated using the medium salt buffer (Buffer A + 200mM NaCl) and high salt buffer (Buffer A + 500mM NaCl). The spectrophotometer was blanked with each buffer before the readings of its corresponding fractions were obtained. The fractions with the highest OD reading at 280nm was collected in an Eppendorf tube, labeled, and placed on ice. The column was then washed with 10mL resin cleaning buffer which was discarded in a wash beaker. Protein Electrophoresis 30uL of 3X Sample Buffer (bromophenol blue, glycerol, dithiothreitol, and SDS) was added to 60ul of each of the 9 samples (standard, homogenate, Pellet I, Pellet I low, medium and high salt buffers, Pellet II low, medium and high salt buffers). The 9 tubes were heated in a water bath for 4 minutes. Into a prepared buffer chamber, approximately 20uL of each sample were loaded into separate wells using thin-barrel pipette tips. The gel was run at 180 Volts for approximately 50 minutes. The gel was removed from the glass plate sandwich and stained in Staining/Fixing Solution for 30 minutes. The gel was destained overnight in Destaining Solution and then dried ona vacuum gel dryer for one hour. Results Table 1: ODwavelength Readings of Protein Fractions for Pellet I OD Reading (280nm) Tube Low Salt Medium Salt High Salt 1 0.202 -0.077 0.020 2 0.162 0.022 -0.083 3 -0.015 -0.077 -0.059 4 0.246 0.003 -0.068 5 0.002 0.020 0.052 OD readings (280nm) for the fractions were measured using a spectrophotometer after they eluted from the ion exchange column. Table 2: OD Readings of Protein Fractions for Pellet II OD Reading (280nm) Tube Low Salt Medium Salt High Salt 1 HI 0.121 -0.069 2 HI 0.687 -0.059 3 1.442 -0.017 -0.095 4 0.963 0.025 0.200 5 0.229 -0.049 0.320 OD readings (280nm) for the fractions were measured using a spectrophotometer after they eluted from the ion exchange column. Table 3: Known Protein Standards, Relative Molecular Weights and Distance Traveled on Gel Proteins Distance Traveled (mm) Molecular Weight (Daltons) Phosphorylase b 80 97,000 Albumin 130 66,000 Ovalbumin 200 45,000 Carbonic anhydrase 260 30,000 Trypsin Indicator 20,100 Lactalbumin 14,400 The molecular weights of the proteins are known and the distance traveled was gotten by measuring with a ruler from the top of the well to the band of each band. Weight (kDa) Solutions of proteins with known weights were loaded and run in SDS-PAGE and the distance was measured from the top of the well to the bottom of the band. Table 4: title Sample # Sample No. of bands Migration distance (cm) Molecular Weight (kDa) 1 Pellet I Low salt 2 PI Medium salt 3 PI High salt 4 PI 1 2.7 28 5 Pellet II Low salt 1 2.8 26 6 PII Medium salt 7 PII High salt 8 Standard 1 2 3 4 0.8 1.3 2 2.6 97 66 45 30 9 Homogenate 1 2 3 1.8 2.3 2.6 50.3 36 30 The number of bands were tallied, then the molecular weight was collected as well as the migration distance. The migration was found by measuring from the top of the well to the bottom of the band. Four bands showed in the lane where the standard was loaded instead six, because there were six proteins present in the sample this does not make sense to me. Three bands give sizes appeared in the homogenate and one band in Pellet I and Pellet II low salt. Discussion Rubisco weighs 55,000 kDa according to literature and gel bands that correlate with it that size did not appear in either of the samples. It was predicted Rubisco is very negatively charged and very soluble, therefore it should elute the column at a high salt concentration because a large concentration of salt should be needed to disrupt the bonds created between the negatively charged ion of Rubisco and the positively charged resin, but the high salt buffer fractions had some of the lowest OD readings which is conflicting with our prediction. Instead, the OD readings were highest with the low salt buffer concentration. This could mean that Rubisco is not as negatively charged and soluble as we predicted. I cannot clearly conclude if Rubisco was isolated or not because the gel ripped and a big chunk of it was missing due to mishandling. Also, Rubisco might have been lost due to contamination because they were no bands that correlated with its molecular weight. Good! The quality of the experiment could be improved by some additional methods to identify Rubisco since it fixes carbon dioxide. Therefore they could be a test to show that the protein that was isolated can actually fix carbon dioxide.

An Examination of Power and Politics in Organizations Essay

Abstract Many studies have examined the affects power and politics have on organizations. In researching The University of Phoenix’s Online Library and current web sites information shows that power and politics dominates the work place and organizations more than any other personal skill. In order to lead one must have power to influence, set and enforce rules. Politics are found in the majority of organizations, therefore it is crucial for employees to recognize the political game in their own organization and decide rather to participate and benefit from the positives of politics or not. An Examination of Power and Politics in Organizations Power and politics are dominating forces with in organizations. Organizations are lead by people with power. Power can be awarded by management to a person by title, â€Å"legitimate power† which is more formal and recognizable. Many leaders assume power through such things as knowledge, information or seniority. Through research located online and knowledge obtained from personal experiences a comparison was conducted on power and politics in organizations. The structure of a team is important in determining how the team will perform. Each team member should contribute skills to enhance the team’s overall performance. Individual work habits and leadership styles will determine if the team has the ability to join together and complete their common goals. Teams create environments that bring out the best in each team member. A commitment to effectively communicating information and ideas is a characteristic of an effective team. That is why the usage of coercion and politics to gain advantage over team members can be very destructive to team dynamics. Coercion is defined by the Oxford English Dictionary is to force one to act or think in a certain manner, to dominate, restrain, or control by force. Often at work or in a group environment one might utilize this form of power to control dominance  over the group. This usage of power can often make other team members conform. In a successful team conformity is not a useful tactic. The usage of politics and coercion to gain advantage over team members is a danger to the success of the project. Once team members realize that their input is not wanted more than likely they will withdraw from the process altogether. The USA today states, â€Å"offices dynamics often change from the usage of intimidation†. (2004) For example a team must work together to process information for the desired results. Once a member takes advantage of a situation and use coercion to gain advantage it often leads to disaster. An example of such disaster happened recently within our team. A couple classes ago we had a member that was an extremely dominant person. The person was very smart and well educated but had a problem understanding the syllabus. Everybody in the team would understand the group assignments one way but she would always play devil advocate on the simplest details. She would use coercion to gain advantage over the other team members when we would meet to complete group assignments. The entire team was guilty of just going with the flow, not wanting to cause any problem. She was so convincing we felt she had to be correct! The result was a low grade, because the content was not correct. So the team from that moment on did not allow that dominant personality use politics and power to manipulate the group any more. Organizational Behavior Ch. 15 pg. 3 defines power through information as; Information power is the access to and/or the control of information. It is one of the most important aspects of legitimacy. In the majority organization the person with the information controls the manner in which the company operates. Expert power is the ability to control through the possession of knowledge, experience, or judgment that the other person does not have but needs. This source of power is often used by subordinates. Judson, P. stated in Worldwide Energy â€Å"although there are several directors and managers to organizations the experts of the business actually controls progress.† In the public works and engineering department there are several directors and managers. Hierarchical authority has put several rules, guide lines and manuals together through information they obtain for employees to  follow when completing tasks and goals. Supervisors and employees who actually work in the field have the knowledge and experience; therefore tasks are completed in a different manner than the manuals implement. The knowledge subordinates obtain through actually doing the work gives them more power and control in decision made about the organization. Politics are in all organizations and employees are affected in some way by politics. As stated in by Schermerhorn, Hunt and Osborn, â€Å"any study of power and influence inevitably leads to the subject of â€Å"politics.† â€Å"For many, this word may conjure up thoughts of illicit deals, favors and special personal relationships.† Although these actions are considered negative by the observer, the organization and the recipients view politics as positive. Organizations use politics to influence employees through promotions and rewards. The City of Houston Public Works and Engineering Department mainly promotes from with in the organization. This is a positive showing by management, because employees feel effort are rewarded. When you think of power and politics rarely are these images positive or inspiring. Corporate American today has fallen far from the one time logic that rewards are based simply on individual performance. Today more rewards are based on the managements need for power. This ability, power, to have someone else do something in the way you want it done and the precise time you need it done is an overwhelming and influential force. There are several types of ruling powers that are used in today’s businesses. Perhaps, the most preferred by employees is known as the Reward Power. The Reward Power enables a manager to express their appreciation to an individual or group for a job well done by offering the employee or team members a bonus. Such pleasantries can be in the form of a pay increase, personal praise, or advancement within the company. Unlike the Coercive Reward, probably the most disliked by associates, is a system in which the person in charge uses punishment to rule. Examples of such authority could be the withholding of merit increases, denying a promotion, or even termination. Powers such as these are key elements to leadership success. Unfortunately, they seem to represent the seamy side of management (Schermerhorn, 2003). There are other powers used in organizational structures, such as legitimate, process,  informational and representative. These power all vary in pattern, however, none are as contrasting as the Reward versus Coercive Power. How do these managers gain this power? Some might believe that office politics and networking are major components. The saying â€Å"it’s not what you know, but who†, does not stand truer than in today’s corporate environments. An example of â€Å"networking† took place a couple of years ago in our department. At the time of this occurrence, Sam was a Financial Administrator for the West Region. He held a reputation for one of the best managed financial divisions. It seems that Sam had built a special friendship with the Sales manager for that region and he would do allow special payments or exceptions for his market without proper documentation to help his numbers increase. These ‘favors’ were soon shared with other salespeople from other markets, and Sam immediately became best of buddies with everyone. A few months later a position for a supervisor was opened and Sam immediately recruited his comrades to assist him in assuring this new posi tion. His use of political power and networking is a prime example of the self absorbed need for dominance in today’s managers. Ethical Persuasion and Legitimate Power can have a strong impact on teams, management and others alike. First, let’s look at the definition of ethical persuasion and legitimate power. Ethical persuasion is behavior that is morally accepted as â€Å"good† and â€Å"right† (Schermerhorn, gloss, pg. 6). Legitimate power is the extent to which a manager can use the â€Å"right of command† to control other people (Schermerhorn, gloss, pg.12). Ethical Persuasion is very different from legitimate power because when a manger is demanding that you complete a task, the task may not always be ethical. Each one of these behaviors can affect a team in many ways. For example, a management team is assigned a task that involves investigating an employee’s work history to locate any negative infractions or practices in order to terminate the employee. In the investigating process you learn that this person is a single-mother barely able to feed her two children. However, the company cannot afford to keep her for financial reasons and needs to lay her off. There are five members in the management team that are working on this case and three of them are females, these ladies do not feel that they are  displaying ethical behavior and refuse to have anything to do with dismissing this single mother. In reality this woman has done nothing wrong, she was just the last one hired. In the meantime the CEO hears about the moral conflict and he gets very upset with the team members disobedience. Consequently, the CEO calls a meeting to let the management team know that he is the head of the company and team will do as he says whether they like it or not. If the team do not do as they are told, then they can give the single mother their positions because they will no longer be needed. In this situation the CEO is clearly using legitimate power, which is definitely not ethical; however, it is a job that still has to be done. Therefore, ethical behavior and legitimate power can and will eventually affect a management team. More often than not legitimate power will overrule ethical behavior. In conclusion research has proven sources of position and personal power, such as expert, information, reward, coercion and legitimate are used by employees to control, persuade and influence people and organizations. Politics are more commonly used by management to control and influence organizational behavior. Both politics and power have positive and negative affects on employees, organizations and communities. References Lindsey, L. B. (2004, July 14) Office Dynamic; Retrieved August 26, 2004 from University of Phoenix, EDSCO Host database, Website http://phoenix.edu//libraryresource Stum, D.L. Strategy and Leadership. Maslow Revisited. Building the Employee Commitment Pyramid Volume 29. University of Phoenix Custom Edition. Retrieved August 25, 2004, from ProQuest database. Judson, P. (2004) Advantages of Experts; Worldwide Energy. Vol. 15 Issue 8 p3. abstract retrieved August 28, 2004, from EBSCOhost data base Schermerhorn, J.R., Hunt, J.G. & Osburn, R.N. (2003) Organizational Behavior New York: John Wiley & Sons, Inc. Ch. 15 Power and Politics Pg 2-3

City Of God Movie Review The City Of God - 817 Words

The movie, The City of God, is based on a true story that took place during the early 1960s in Rio de Janeiro, Brazil. The movie tells a story from the point of view of a boy named Rocket. He was born and raised in the slums of Rio de Janeiro. Throughout the film, he struggles with the fear of being dragged into crime, violence, and drugs. His goal is to one day break free from the slums in which he is currently trapped. The surrounding environment and society during this time was highly influenced by what everyone else was doing. The main characters were saboteurs and always causing corruption around town. Some notable characters would be Shaggy, Goose, and Clipper who together comprised a group called the â€Å"Tender Trio.† They were†¦show more content†¦It allows the audience to connect with each individual character and to understand the characters’ feelings. One scene in this film that had a big impact on me related to a â€Å"runt† named, Steak. Steak classified himself as a man because he said he kills, snorts, and smokes. The word â€Å"runt† in this film is another name for a young kid. He was only about 8 years old. The scene depicts Steak bragging to superior people in order to fit in. He felt peer pressured to say he did all these horrible acts, which was extremely saddening because, in reality, a kid his age should never be exposed to crime, murder, or drugs. During this time Brazil is a dictatorship but the film does not emphasize it enough to make it a central theme of the movie. It is a shame that kids who grow up in these certain situations don’t know any other way of life than the type of culture they are surrounded by. The idea of little kids killing people just perpetuates the trend of senseless killing that seems to be prominent in these communities. Life does not seem to retain much value to them. It becomes their nature to kill and do drugs, starting at such a young age. In a very similar way, kids in more developed communities, like America, do not fully understand communities where every day is a fight for life. Whether it was decades ago or today, it is crazy to think that horrific situations like these occur at all. Killings, armed robberies, drugs, and crime, defines life in the slums.Show MoreRelatedA Theological Reflection On The Dark Knight Rises1249 Words   |  5 PagesWrite a theological reflection on a recent movie of your choice In one or two paragraphs, give a brief summary of the plot and its primary theme or main point (approx. 10% of the total essay) For this assignment I have chosen to review The Dark Knight Rises. Eight years after the Joker s reign of anarchy, the Dark Knight is forced to return from his imposed exile to save Gotham City from the brutal guerrilla terrorist Bane with the help of the enigmatic Catwoman. 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Climate Change And Its Effect On The Environment - 2241 Words

Introduction The past two decades has see the largest push for the creation renewable energy technologies in order to reduce the anthropogenic carbon emissions, that have risen so quickly since the industrial revolution began in the early 19th century. The common use of cars, the expansion of commercial farming and the large use of everyday use of fossil fuels such as coal, oil and gas have all contributed to the rising levels of green house gasses. Carbon dioxide levels have increased by around 38% and methane levels have increased by 148% (Earthobservatory.nasa.gov, n.d.) both carbon dioxide and methane are considered to be green house gasses. The warming of the earth’s climate has gone hand in hand with the rising green house gas†¦show more content†¦In renewable technologies such as wind turbines the most prominent REEs are neodymium (Nd), Praseodymium (Pr) and dysprosium (Dy). 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Scotland is abundant in the natural resources need to create efficient renewable energies. These are mainly in the form of on land wind farms closely followed by off shore wind farms, but it is also very involved in designing efficient wave and tidal energy forms. The majority of Scotland is rural in its landscape meaning that in a lot of areas the onlyShow MoreRelatedThe Effects Of Climate Change On The Environment Essay1268 Words   |  6 PagesOCEAN ACIDIFICATION Smog, contaminated water, melting of snow packs. These are some of impacts that Climate Change has had on the Earth over the past years. All of these are product of the increase in carbon dioxide emissions by humans and their polluting activities. Since the Industrial Revolution, these events have escalated and, they have been becoming more noticeable and prolonged across the globe. Some of these events are more palpable and pronounced in certain parts of the hemisphere. 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