Cellular Fractionation | Lab Report

Cellular Fractionation | Lab Report Performer: Aylin NEBOL Partners name: Dila ERDEM INTRODUCTION Centrifuge is a laboratory device that spins objects which are put inside it in a fixed rotation with very high speeds and uses centrifugal force to move the objects outward. In centrifugation process two different measurement units are used: RPM and RCF. Revolutions Per Minute (RPM) is the measurement of how many revolutions the centrifuge completes in one minute. Simply, RPM value tells the rotors speed of spinning. The Relative Centrifugal Force (RCF) is the measurement of gravitational force that the rotation speed of the centrifuge applies on a sample an it is expressed in the unit of gravity (Difference Between RCF and RPM in Centrifugation n.d.). With the centrifugation process, denser particles accumulate away from the rotation centre and the lighter ones towards the centre. The denser ones are known as pellet and the remaining solution is supernatant (Journal of visualized experiments: JoVE. n.d.). Differential centrifugation is a procedure that is used to deeply analyse certain organelles by separating them from others considering their size and density. In this technique, particles with higher density travel toward the bottom of the centrifuge tube at a faster rate. (Centrifugation Biology-Online Dictionary 2005). During cellular fractionation, the temperature should be under control. To make centrifugation and fractionation effectively temperature must be kept around 4 degree Celsius to protect DNA and proteins from degradation. When centrifuge machine is started, the observer should not be leave there until maximum speed is reached in case of any possible problem like that machine may be stop. The other factor is choosing the most appropriate tissue. Tissues should be chosen based on their organelles which are wanted to be seen and examined at the end of the centrifugation and suspension processes. Homogenization is a process in that cells are opened in an isotonic buffer to isolate different organelles from cells. Various types of homogenization can be applied to substances. Sonication is a way that uses the high frequency sound waves to break open cells. Detergent lysis is to use chemicals, French press breaks open cells by applying high pressure and mechanical homogenization is the way that uses a high-speed blender to break open cell. There are two main types of centrifuge rotors: Fixed angled and swinging bucket. At fixed angled rotor pellet accumulates on the walls of the tube while it accumulates at the bottom when centrifugated with swinging bucket. Since the centrifuge works with centrifugal force change in speed, gravitational force and frictional resistance affects the content of the pellet. Since rotor radius has an effect on RPM and RCF values, it also effects the centrifugation (Katkov and Mazur 1999). AIM The aim on this experiment was to obtain mitochondrial pellet through gradually cellular fractionation process with centrifugation machine. Besides, the aim was to observe how to homogenize and centrifugate samples, also the difference between different rotor types and their function. MATERIALS Chemicals: Fresh rat liver Solutions: 0.25M sucrose Lab Equipment: 50 ml and 15 ml centrifuge tubes Ice bucket Homogenizer Table top and high speed centrifuges Vortex Scale Maxi-pipette/ Serological Pipette Bulb Pasteur pipette METHODS Preparation of sample and Homogenization: 10 ml of 0.25M Sucrose was poured onto 1 g rat liver that was taken from ice bucket. Prepared mixture was homogenized with blender, starting with low speed and increased. Sample was labelled. The First Centrifugation. The homogenate was taken to table-top centrifuge working with 800 g (2037 RPM) force and centrifugated for 5 minutes at 4 degree Celsius. This centrifugation was done with the swinging-bucket model rotors whose model name was SX 4250. After centrifugation, supernatant was separated from pellet clearly by pouring it to another 15-ml centrifuge tube then supernatant was kept and labelled, and pellet parts were thrown away. In the end of the procedure we got cell debris, cell membrane and cytoskeleton. The Second Centrifugation: The second centrifugation was made with a different centrifuge (J2 221) which was worked with 5000 g (5710 RPM) speed and fixed angle rotors whose model name was JA-14. Precision scale was used to balance the masses. The masses placed carefully and symmetrically. The samples were centrifugated for 15 minutes and again at 4oC. After centrifugation supernatant was kept and labelled. While separating supernatant from pellet it poured into a 15ml centrifuge tube from the opposite side of the pellet. In the end of the procedure nuclear pellet was seen. The Third Centrifugation: This centrifugation step was made with the same centrifuge with the 2. centrifugation: J2-21 model Beckmann and JA14 model fixed angle rotor. To balance the opposite weights, sucrose was added to some empty tubes on the adaptors. After balance was achieved, adaptors were put oppositely in the centrifuge. The inside cover was closed. Sample rotated with 24.000 g (12512 RPM), for 10 minutes and the temperature was set between 4 and 10 degree Celsius. After centrifugation supernatant was thrown away and the pellet was kept this time. Supernatant and pellet were separated with serological pipette and three-way bulb. The pipette was hold on the opposite side of the pellet in a way that it did not touch the pellet. In the remaining part without supernatant, existence of mitochondrial pellet was recorded. Resuspension of the mitochondrial pellet: 5 ml 0.25 M Sucrose was added onto the mitochondrial pellet. Maxi pipette was used to dissolve the pellet in the sucrose by pipetting up and down without touching the pellet and taking liquid part from the opposite wall of tube according to pellet. The mixture was stirred clearly with vortex. Mitochondrial suspension was obtained. RESULTS 1 gram rat liver was homogenized with 10ml of 0.25M sucrose in order to separate cell parts and organelles with centrifuge properly. In each step the sample was placed on ice rather than being waited at room temperature to inhibit protein degradation. Similarly, in centrifugation part of the experiment to prevent protein deformation temperature was kept around 4oC. Masses on the adaptors were balanced with some tubes with sucrose to prevent any error resulting from asymmetrically placed masses on the adaptors. Moreover, to prevent any centrifuge related problem the centrifuge was waited to reach its maximum speed that is desired for the experiment. The sample was homogenized successfully by changing its speed. After the first centrifugation with table top centrifuge working with 800g RCF and 2037 RPM values, where swinging bucket rotor was used, cell debris, cell membrane and cytoskeleton were seen in the pellet. The radius of the centrifuge was calculated as 17.2 cm. The second centrifugation was done by using a fixed angle rotor at 5000 g RCF value which corresponds to 5710 RPM value. After this step, pellet was gathered not on the bottom of the tube as the first one but seen on the wall. The colour was darker on the bottom and lighter on top. Nuclear pellet was observed. The third centrifugation was done by using fixed angle rotor at 24000g that corresponds to 12512 RPM value. After this step, supernatant and pellet were separated with serological pipette and three-way bulb. In the remaining part, mitochondrial pellet was observed. At the final step, the remaining pellet was suspended by adding sucrose and stirring the mixture gently until obtain a clear mitochondrial suspension. DISCUSSION The aim of this experiment was to observe not only homogenization and centrifugation processes but also how to take care of homogenizer and centrifuge. Centrifugation process was applied to the sample step by step till desired molecules were obtained. In this experiment, last step was to get mitochondrial pellet. Since the purpose of the experiment was to obtain mitochondrial pellet, a structure that is mitochondrially rich should have been chosen. Liver cells are responsible for many metabolic activity resulting in needing high amount of energy to continue the process and finally abundance of mitochondria. Which is why the liver cells were the best samples to examine for cellular fractionation. (Caprette 2012) Sucrose is a buffer that is isotonic at 0.25M and does not hinder enzymes activity in animal tissues also not react chemically with organelles so prevents the mitochondria lysis during the process. Those characteristics make Sucrose one of the most useful buffers, since isotonic buffers are used to homogenize tissues appropriately. Also, the density and size of sucrose molecules make it easy to suspend pellets, balance centrifugation and even pour off it as supernatant at the end of each centrifugation. (Clayton and Shadel 2014) All the centrifugation steps were carried out at low temperatures to adjust the heat revealed by friction force against the rotating speed of the centrifuge. Organelles were stored at -70 °C to inhibit the cellular damage and inactivate the enzymes that lysis the cells. In the second centrifugation, in the instruction manual the RPM value for JA-14 model fixed angle rotor was given as in between 5500-6000 values for 500g RCF and 13.7 cm radius. Yet, from the RCF = 1.119 X 10-5 (RPM)2 X r equation, the RPM value was calculated as 5710. In instruction manual, the RPM value, similarly, was given as 12500 since same rotor model was used. Yet according to our calculations the RPM value was 12512. Totally pure organelle fractions cannot be obtained with differential fractionation since this method separates organelles based on their size and density. Thus, it is necessary to use another method that separate samples considering their density. This method is density- gradient centrifugation. It should be centrifugate samples at high speeds and for hours to let each cellular component to migrate their equilibrium positions (Lodish et al. 2000). REFERENCES Caprette, R. David. 2012. Tissue Fractionation. http://www.ruf.rice.edu/~bioslabs/methods/fractionation/fractionation.html (March 14, 2017). Centrifugation Biology-Online Dictionary. 2005. http://www.biology-online.org/dictionary/Centrifugation (March 10, 2017). Clayton, D. A., and G. S. Shadel. 2014. Isolation of Mitochondria from Cells and Tissues. Cold Spring Harbor Protocols 2014(10): pdb.top074542-top074542. http://www.cshprotocols.org/cgi/doi/10.1101/pdb.top074542 (March 14, 2017). Difference Between RCF and RPM in Centrifugation. http://www.westlab.com.au/page/97/Difference_Between_RCF_and_RPM_in_Centrifugation.html (March 10, 2017). Journal of Visualized Experimentsà ¢Ã¢â€š ¬Ã‚ ¯: JoVE. https://www.jove.com/science-education/5019/an-introduction-to-the-centrifuge (March 10, 2017). Katkov, Igor I., and Peter Mazur. 1999. Factors Affecting Yield and Survival of Cells When Suspensions Are Subjected to Centrifugation. Cell Biochemistry and Biophysics 31(3): 231-45. http://www.ncbi.nlm.nih.gov/pubmed/10736749 (March 15, 2017). Lodish, Harvey et al. 2000. Purification of Cells and Their Parts. https://www.ncbi.nlm.nih.gov/books/NBK21492/ (March 15, 2017). APPENDICES Calculation of radius of the first centrifuge: RCF = 1.119 X 10-5 (RPM)2 X r 800 g = 1.119 X 10-5 (2037)2 X r r = 17.2 cm Calculation of RPM values: RCF = 1.119 X 10-5 (RPM)2 X r No.2 centrifuge: 5000 g = 1.119 X 10-5 (RPM)2 X 13.7 RPM = 5710 No.3 centrifuge: 24000 g = 1.119 X 10-5 (RPM)2 X 13.7 RPM = 12512      

“Hot” Spots in the Cold War Essay

The period of the Cold War has turned into the dramatic stage of fight between the two world’s largest powers – the United States and the U. S. S. R. The Cold War remains a remarkable moment for the U. S. , when the majority of the American people could check their strength and endurance. It did not matter whether the U. S. was promoting the politics of Soviet Containment, or was involved into Cuban Missile Crisis, whether the U. S. was flooded with McCarthyism ideas, or was building Levittown to restore the American dream – all those actions were later included into the set of events under the general title The Cold War. â€Å"Containment of the Soviet Union became American policy in the postwar years† (Stanley 11). It is interesting to note, that while the U. S. was fighting against fascism and took the Soviet Union as its ally, all anti-communism ambitions were neutralized or simply muted. The Cold War has started immediately after the end of the WWII and raised numerous issues in the political relations between the two super-states. The idea of political containment belonged to George Kennan: in his article published in 1946, he discussed and evaluated the sense of insecurity the U. S. S. R. traditionally experienced (Stanley 8). As a result, the discussed insecurity sense made it possible for Kennan to suggest that â€Å"Moscow’s pressure to expand its power had to be stopped through â€Å"firm and vigilant containment of Russian expansive tendencies† (Stanley 18). On the one hand, the policy of Soviet Containment supported by Truman was the means to protect the U. S. territory from external military threats. On the other hand, Truman has evidently overstated the threat coming from the Soviet Union. As a result, the policy of containment has generated mass anti-communism which sometimes bordered with hysteria. Consequentially, the United States containment policies gave rise to the well-known McCarthyism and were also displayed in the U. S. involvement into the Cuban Missile Crisis. â€Å"McCarthyism is a term describing the intense anti-communist suspicion in the United States in the period that lasted roughly from the late 1940s to the late 1950s† (Stanley 24). This term has for long been connected with the name of Senator Joseph McCarthy, but it has currently expanded its meaning to more general definition of anti-communist policies in the U. S. Joseph McCarthy has actually been the initiator of the discussed policy which later turned into the whole cultural phenomenon. In his speech on February 9, 1950, he announced that he had â€Å"a list of 205 – a list of names that were made known to the Secretary of State as being members of the Communist Party† (Stanley 25). McCarthy’s speech and ideas were later reflected in the invention of loyalty-security reviews, blacklists, and even arrests. As a result of mass anti-communist campaigns, twelve thousand people lost their jobs without any hope to find new employment: negative loyalty reviews made it impossible for a person to become employed (Zinn 411). In reality, such policies could eventually turn into a â€Å"hot war† and have involved the United States into the actions which made the threat of the nuclear war as close as never before. The realization came with the events which were later called The Cuban Missile Crisis. The Cuban Missile Crisis was one of the critical hotspots in the history of the Cold War. Its origins were rooted in both Containment and McCarthyism policies, and supported the line of opposition towards the Soviet power. In 1962 the Soviet Union was searching the means of strengthening its position on the international political arena. The Soviets could achieve that aim only by strengthening their military potential (Stanley 32). As Khrushev wanted to place the Soviet missiles in Cuba, Fidel Castro was looking for the ways of protecting his country from the U. S. intervention. However, Kennedy was aware of the Soviet and Cuban plans: â€Å"On October 22, Kennedy announced the discovery of the missile installations to the public and his decision to quarantine the island. He also proclaimed that any nuclear missile launched from Cuba would be regarded as an attack on the United States by the Soviet Union† (Stanley 33). In order to eliminate the threat of military conflicts between the U. S. and Cuba, the Soviet Union had to refuse from the idea to locate their missiles on the Cuban territory. As the country found itself in the whirl of political and military conflicts, the common people had to restore the country after the WWII, and had to prove to themselves that they were able to survive the difficulties of the Cold War. Levittown has become the brightest display of how people were trying to improve their living even under the pressure of McCarthyism and Containment. In 1950s, William Levitt actually created the prototype of a middle-class American suburb: small, convenient and cozy houses for medium families have attracted attention of numerous architects and economists. By the time the town was completed, it included buildings, swimming-pools, schools, churches, 17,000 single-family houses, and occupied 5500 acres of Bucks County in Pennsylvania (Zinn 407). However, the historical meaning of Levittown was not in the way it was constructed, but in the time period during which it was created. Levittown has given the rise to creating affordable housing and has served the pattern of non-compliance with the principles of the Cold War. In his town, Levitt was able to â€Å"present a new form of ideal American life, one that combined the idealized middle-class life of the prewar suburban communities, with the democratized life of younger GIs and their families† (Zinn 409). Works Cited Stanley, George Edward. America and the Cold War (1949-1969). A Primary Source of History of the United States. World Almanac Books, 2005. Zinn, H. People’s History of the United States: 1492 to Present. Harper Perennial modern Classics, 2005.

Get the Scoop on Introduction of Argumentative Essay Samples Before Youre Too Late

Get the Scoop on Introduction of Argumentative Essay Samples Before You're Too Late You might also see concept essays. So you were assigned with a persuasive essay but you need no clue what it appears like. In order to give an in-depth understanding about the argumentative essay, it is advisable to take a look at some of the greatest examples of argumentative essay. A very good persuasive essay example should show you your thesis statement should accomplish three things. To begin with, you will want to understand what an argumentative essay is. First of all, you need to understand what an argumentative essay is. Every argumentative essay ought to be based on a topic that may be debated. It is very important to be aware an argumentative essay and an expository essay could be similar, but they vary greatly with regard to the quantity of pre-writing and research involved. The Most Popular Introduction of Argumentative Essay Samples A decent persuasive argument will use the latest data and data from verified sources. Though your introduction has to be short, it also ought to convey plenty of information. How much space you will need for background is dependent on your topic and the range of your essay. You should introduce your precise topic and offer any essential background information that the reader would have to have in order to comprehend the problem that you're presenting in the paper. You might also want to incorporate a quick discussion of more research that ought to be completed in light of your work. Science experiments are among the most authentic methods of getting knowledge about the world around. Choose from popular topics that folks are passionate about. Third, students will be mindful of the risks and negative effects that could emerge in the event of improper sexual behavior. Introduction of Argumentative Essay Samples - Is it a Scam? Any writing work demands an impeccable introduction so as to transition to the succeeding portions of the paper. If you need assistance, you could always find someone who's writing essays for college cheap. Tell our experts what sort of homework help on the internet you will need to get. Finding out how to compose an essay introduction may appear the very best start for virtually any student who is fighting with his academic endeavor. Argumentative essays utilize logic, facts, and reasoning to set the victor. Rem ember an argumentative essay is based more on facts rather than emotion. Bear in mind a persuasive essay is simply a reflection of your beliefs or opinions. Any fantastic task finishes with a wonderful conclusion and the very best examples of the argumentative essay will arrive in with a conclusion which has an overview of all of the points together with a gist of the evidences provided. What's Actually Going on with Introduction of Argumentative Essay Samples In many instances, you can move straight from your introduction to the very first paragraph of your physique. Bear in mind that it's not essential to compose the introduction first. In the end, the introduction must conclude with a very clear statement of the total point you wish to make in the paper. It has three essential parts, each of which serves a particular purpose. If You Read Nothing Else Today, Read This Report on Introduction of Argumentative Essay Samples The very first sentence of the essay has to be int eresting enough to produce the reader read on. Probably you do, because you're hunting for assistance with your essay writing. Bear in mind that the period of your essay is based on the assignment provided to you. A five-paragraph essay may be as long as 2000 words and you have to keep an eye about the quantity of words in 1 page. Possessing the most suitable vocabulary is critical for writing a first-class essay. When you're writing an abortion argumentative essay, you're absolutely free to support any side that you desire. It isn't a must, but should you care about your topic, it's going to be resembled in your writing. You might also locate the precise subject of your writing. An argumentative essay is a sort of written assignment where the author describes an issue or a circumstance and supplies the examples of two contrasting opinions on this issue. It has a specific format that needs to be followed to blow the mind of the reader, and it is especially useful for students as well as the corporate while making strategic proposals. Writing an argumentative essay can occasionally be confusing since you don't necessarily understand how to compose a convincing argument. Writing a persuasive, argumentative essay can be challenging, and at times it can find a little confusing.

Managing Organizational Structure at Apple Inc - 1521 Words

Introduction Apple Inc. was established by Steve Jobs and Steve Wozniak on April 1, 1976 as a computer designer, developer and seller company. However, the company shifted its focus from only personal computer to include other consumer electronics such as portable media player and mobile phone in 2007. Apple Inc becomes one of the most popular makers in its field since it seems that its popularity has increased according to a report on www.statista.com that Apple Inc’s products sales was generally increasing throughout the first quarter of 2006 to the first quarter of 2014. On the one hand, it has increased its revenue from about 14 billion US dollars to more than 170 billion US dollars in 2013. All in all, the company is highly†¦show more content†¦Organisational design decisions Mechanistic or organic organisation Apple Inc can be considered to be more mechanistic organisation than organic one. It is high specialisation; employees are working based on their skills. The company has quite rigid departmentalisation as it consists of 15 different departments: finance, legal, software engineering, marketing, retail and online stores, hardware engineering, industrial design and human interface, internet software and maps, operations, special projects, sale and service, Apple university, human resources, international sales and North American sales. And these departments have over 40 sections in total under their controls. Apple Inc is managed through chain of command, information flow from the top to bottom line of command. This company is fairly high formalisation with some strict rules and procedures. It is also centralised company as most decisions will be made from upper level management. However, the company has wide span of control which is a characteristic of organic organisation as there are high number of employees under managers in the chain of command, over 80,000 worldwide employees are in charged by 15 departments, over 40 sections. 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