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.

In the Shooting Salvationist, David R. Stokes recounts the murder Essay

In the Shooting Salvationist, David R. Stokes recounts the murder trial of fundamentalist clergyman J. Frank Norris. Despite muc - Essay Example The court was told of the Norris’ feuds with the Fort Worth Mayor where the preacher had alleged that the city Mayor was an adulterer. Mayor Meacham swore to his friend, D.E Chipps that, â€Å"something must be done† to stop Norris behavior (Stokes 56). Chipps, a local businessperson, agreed to face up to the preacher to threaten him against tainting the local politicians. On July 17, 1926, Chipps entered Norris office and threatened to kill him if he continued to attack the Mayor. According to an eyewitness, L.H. Nutt, Chipps then appeared to have left the building before storming back with the same threats. Norris was standing in front of his desk where a gun belonging to the guard of the church was kept in a drawer as a ‘safety precaution’. Norris then short Chipps three times and claiming that the attacker was â€Å"stronger and could easily have overpowered† him had the brawl resulted into a physical fight (Stokes 112). The defense team stood it s ground that Norris acted in self-defense. According to Stokes, the defense team insisted that Norris’ popularity, fundamentalism views on modernity and his castigation of local leaders and sinners had attracted many enemies including the Mayor. Stokes and the final verdict seemed to agree that Dr. Frank Norris had not premeditated to murder Chipps. He only feared that the drunkard man endangered his life. To strengthen their case further, the defense team presented evidence of Norris inquiring from Nutt about Chipps identity. The case would have yielded different results had the court been presented with evidence that the preacher indeed had prior acquaintance with the victim. On these grounds, the court appeared to be reluctant to convict Dr. Norris of first-degree murder (Stokes, 34). Previous acquittal of Dr. Norris on arson and perjury also served to strengthen his ‘not-guilty’ plea. The prosecution team tried unsuccessfully to relate the two cases as proof that the preacher was culpable of the murder. This was never going to add weight to the case that was facing public scrutiny like never before in the American justice delivery history. There was an apparent fall-out between the public and the once renowned preacher. This was a weak point in the prosecution team because of the enormous task to proof behind reasonable doubt that it was not acting due to public pressure. Ultimately, this proved to be the breakdown of the trial in Norris favor as prosecution team appeared to be overreached. Stokes blamed both the jury and the prosecution team for the apparent injustice of finding Norris not guilty. The prosecutors could easily have won the case had they charged Norris with second-degree murder. Their overly vicious immovable decision to stage a â€Å"first-degree murder case or nothing† was not justifiable under the overwhelming evidence of the contrary (Stokes 150). The jury on the other hand appeared not to consider the possib ility of advising the prosecutors to reconsider their stand. Their final decision baffled the whole world because the enormity of the case warranted a better rationale for a ‘not-guilty’ verdict. Stokes noted that Norris had â€Å"great gifts and abilities† but he exercised â€Å"coercion, control and manipulation†¦Ã¢â‚¬  to further his cause (Stokes 105). Dr. Norris strongly believed that â€Å"the end always justified the means† as he went to every length, including lying, to expand his doctrine of

Policy papr Essay Example | Topics and Well Written Essays - 500 words

Policy papr - Essay Example At the end of it all, one can die very quickly as a result of the diseases. Therefore, a policy to govern the levels of consumption of the junk foods has to be applied in order to ensure that people feed on nutritious foods. One of the effective policies that are effective is that junk foods must be taxed to reduce their consumption. Taxation will make the foods expensive than the nutritious foods. As such, people definitely opt to purchasing nutritious foods, which even if consumed in small quantities, lead to satisfaction. Agreeably, junk foods are cheap to acquire and readily available to the consumers (Matz, 2004). This makes it easy for the low-income earners to acquire them because they cannot afford purchasing nutritious foods. By having the prices of junk foods very high through taxation, positively, obesity will be addressed and decrease at a high percentage of about 15 percent. However, not only should the food be taxed, but also be highly taxed to ensure that the prices rise. However, this will hinder the low earners from purchasing it but for those who can afford, they will continue consuming it. Taxation of junk food will trigger so many factors that will lead to low consumption of calories. When the prices are high, agri-business will be encouraged and there will come up new types of snacks, which contain low levels of calorie. As a result of these new snacks, fattening will reduce at a very high considerable rate. This will prevent the consumers from the bad eating habits of consuming lots of calories and sugar. Through taxation, people will be in a position to consume foods that are affordable and contain low amounts of sugar and fats. Since foods that are less starchy will be affordable to all people including those with low capabilities, their consumption will be high and reasonable because they are healthy (Matz,

War And Civilization Assignment Example | Topics and Well Written Essays - 500 words

War And Civilization - Assignment Example Wars have been the violent catalyst for changes throughout history. Wars are, however, part of the agencies that help bring changes and progress in human life and the world. It might sound absurd, but wars are part of the human life activities. Wars simulate national growth, advancement in information technology, medical research, and solve some of the most complex political differences. They evoke bravery and heroism among communities and nations. In medical and health care systems, war has been the subject of appreciation as it has promoted human civilization. It is indeed a catalyst that has brought advances in medical science. Most of the chemical medicines and surgical techniques used in clinics and hospitals were not discovered when people were at peace in their homes. They were discovered and advanced when the need arose particularly when casualties of wars were in dire need of medication. Diseases such as Pestilent that evolved out of the war needed the rapid response with th e appropriate medical treatment (Smith, 2006). There are indeed several contributions of war to human civilization. The US, for example, had economic gains for its people from both World War I and II. If one looks the wars as agents of world peace, they did succeed in promoting peace and keeping the world safe. World War II eliminated Hitler who was a threat to the peace and unity of many people. In the 1973 war, Israel won a battle that enabled it to expand boundaries and acquire land that was previously a forbidden territory.

Critical Analysis Essay Example | Topics and Well Written Essays - 2500 words

Critical Analysis - Essay Example At the same time they aim to deliver specific suggestions through analysis of the research outcomes. The suggestions would be of much benefit to education and in making educational decisions. The article, Motivating boys and motivating girls by Marsh and Martin, supports positivist and anti-positivist research paradigms (Babbie, 2006) to explore the effect of gender as well as other educational constructs on student motivation from different levels of understanding. This combined paradigmatic model on one hand is necessary to dig into the psyche of those who believe in the gender stereotypic model and that of the students who are at the centre of all discussions. On the other hand it would help in accumulating tried and tested factual details of the factors affecting motivation and engagement. While the qualitative or the subjective approach helps in generating meaningful responses from the groups, the quantitative or the objective approach backs the subjective findings by providing with significant data. Depending upon the nature of the research topic, this combined approach is undertaken as any one approach would have been insufficient in critically assessing and evalu ating the validity of the research outcome. A qualitative approach based on interviews and case study of students and teachers gives the research its basic direction. An approximate idea could be formed about the practicality of each model. The authors held interviews with teachers, educational practitioners and students of either gender. The subjective interrogation has been of much help in comprehending the standpoints of the two groups. While the educational practitioners hold the gender stereotypic model functional in student motivation within the class room, the students have been observed to hold neutral views implying the need of good teachers rather than fussing over teacher gender. The subjective approach has actually established the relevance of the

THE ATOMIC BOMB Essay Example | Topics and Well Written Essays - 1250 words

THE ATOMIC BOMB - Essay Example At the end of the study, reasons are provided as to why standing armies’ war cannot be similar to war waged by small armed groups. On the 6th day of August 1945, an American B-29 bomber dropped two atomic bombs in Nagasaki and Hiroshima, bringing the Second World War to an end. The dusk of the war, however introduced a new dawn of controversies regarding deliberate attack of civilians in wars, whether the use of the atomic bomb was necessary, and whether war is a practice only reserved for standing armies and nations as opposed to small armed groups. Concisely, America transformed the style of war on the planet in that prior to the Nagasaki/Hiroshima bombings; civilians had never been portrayed as participants or targets in war. Further debates emerged that the atomic bomb claimed fewer lives than what the actual War could have cost had it been allowed to extend beyond 1945. Owing to the new style of warfare introduced by the atomic bombs’ use, warfare on the planet has taken different paths in which case civilians are now part of wars, terrorism is portrayed as a justifiable practice by some, and war is now split between standing armies and small armed groups. In the light of these, the following discussion text will argue why deliberate attacks on civilians are illegitimate acts of war, that terrorism will never be a justified act, and that the practice of war should remain a preserve of nations and standing armies only; contrary to what the post-Hiroshima/ Nagasaki bombings implied. No definition of war would ever make sense if the terms â€Å"armed† or â€Å"weapons† did not feature anywhere in them. This is because war is a form of struggle against rivals as they strive to exercise victory over the other. As such, war has to be against an armed rival with another armed rival. In the event that one rival is unarmed, whatever struggle ensues thereafter takes an overly different definition from war. It may be

The achievement of civil rights so slow in the period 1954-1957 Essay Example for Free

The achievement of civil rights so slow in the period 1954-1957 Essay Why was the progress towards the achievement of civil rights so slow in the period 1954-1957? From 1954-1957 significant legal progress in the field of civil rights was made, thus instilling confidence in the black community, however due to a number of factors putting these achievements into practice was halted and so civil rights progressed at a slow rate throughout this period of time. In 1954 Brown Vs Topeka was brought before the Supreme Court, with Brown winning the appeal with a unanimous vote. This was a landmark verdict, due to the fact that the judge, Earl Warren had been newly appointed by President Eisenhower and was a Southern Republican, therefore he disobeyed Eisenhower with the decision. Eisenhower was later stated as saying, The biggest damn fool mistake I ever made. Therefore demonstrating the Presidents lack of support for the ruling. It was a triumph for the NAACP who had provided the lawyer Thurgood Marshall and had pushed the case through. It overthrew Plessy Vs Ferguson and so was expected to bring about a dramatic change for black civil rights. Although in principal Brown should have brought about a significant change, the progress from the court decision, to putting desegregation in schools into practice was very slow. This was partly due to the court hearing, as no date was set for when the desegregation of schools must be completed, this resulted in the whites ignoring the court hearing, so maintaining the same ignorant attitude as before. However the court decision had empowered the black community and so Brown II was brought before the courts in 1955, again however this did not result in a definite result. President Eisenhower was also a significant reason for the slow progress of civil rights. When he took over from President Truman, he inherited the beginnings of a strong civil rights campaign, however Eisenhower did not share this same drive. He failed to take substantial leadership towards enforcing the verdict, as he was afraid of stirring up opposition, resentment and disorder in the South. He also did not believe that it was appropriate to interfere in individual state matters, although general consensus suggests that if he had shown strong leadership then the bill would have been put into practice at greater speeds. He believed that race relations would gradually improve on their own accord. Although Brown Vs Topeka overthrew Plessy Vs Ferguson, after the ruling no steps were taken to desegregate public places, again showing the slow progress. However the black community were fed up and so decided to take the matter into their own hands with the Montgomery Bus Boycott. The black community in Montgomery was organised through the NAACP and led by Martin Luther King. Originally they only intended to boycott the buses for a day in order to achieve a more polite service from the bus drivers, employment of black drivers and the end of blacks standing when the bus was not full. However they met opposition from Montgomerys all white officials. This represents why progress was slowed down, as all decisions were met by substantial opposition from whites, forcing any actions to be dramatic, in order to get them to accept their views. This resulted in a yearlong boycott of the Montgomery buses in order to receive total desegregation on the buses. Within this time the White Citizens Councils membership doubled from 6000 to 12,000 from February to March. The boycott attracted national media coverage, showing that the greater publicity that the blacks attracted, the more the whites stuck together in order to create a strong opposition and prevent change. However in this case the blacks economic power was stronger than the white resistance and so in December 1956 the boycott was called off. This shows how much effort it took to achieve progress, however even then it was limited, as the success was only limited to the buses in Montgomery, however it acted as an example for other departments and cities, to what could be achieved through non-violent protests. This was also demonstrated in the case of Emmett Till. When the 14-year-old boy was murdered his mother decided to bring his body home to Chicago and have an open casket. This attracted 100s of the black population that dominated Chicago and the national press to attend. This mass of media coverage, not only united the blacks, and made the moderate whites aware and sympathetic of the cause, but it also ruined any chance of a fair trial. This is because the whites accused the NAACP of using the case as propaganda, so creating a Northern backlash. The southern population then closed ranks, making it harder to prosecute. This again shows that the more publicity that black civil rights was given, the greater the white opposition that they had to face. However this only made the black community more determined. The case of Emmett Till was significant as at first it showed some progress towards a fair trial, with the men accused of lynching arrested, usually they would not have been prosecuted, and a black man standing up and accusing a white man in court. However the jury was composed of 12 white males, and in the defences closing speech he said, I hope every last Anglo-Saxon one of you makes the right decision. Therefore again showing how when accused the whites stuck together. Again throughout this case Eisenhower gave no leadership and did not intervene, as he did not think that federal intervention was appropriate, and did not want to or aim to do anything to alleviate black problems, even when Emmett Tills mother requested his help. Showing again how little progress had been made. Throughout this time, there were strong black leaders, Martin Luther King, and people that The NAACP used for test cases, Rosa Parks and the 9 children in Little Rock. These set examples to the black community, that through courage and determination, slowly change was possible. However many were not willing to make this commitment, like the 16 children who changed their minds about attending Little Rock High School after passing the exam. This was due to the increasing white resistance and discrimination that they faced. Martin Luther King was arrested for doing 30mph in a 25 mph zone, and his house was bombed. As well as this death threats were sent to the childrens houses and they were spat at as they walked down the street. White resistance included the establishment of the Southern Manifesto, which was drafted by Senator Sam Ervin and promised to fight the Brown verdict by legal means. Showing that there was still huge resistance to change, and integration. So making it difficult for progress to continue. As well as this the establishment of the White Citizens Council, which was used as opposition in the Montgomery bus boycott. It was formed to protest the federal government acting dictatorially and seeking to impose its values and opinions on others. This acted as an excuse for Eisenhower, who had little intention of interfering anyway, again slowing down progress. On 3rd of September 1957 9 black children, who had sat and passed an entrance exam, attempted to enter Central High School. However they were met by a hostile crowd of white adults who blocked their way, while shouting abuse such as Nigers go back to the jungle. This was the first significant step towards putting the Brown decision into practice and so attracted a lot of criticism. It signified that the ruling met tremendous grass-roots resistance when put into practice. And so although blacks tried to push segregation, it was clear that it would take a long time for the whites to accept it. On top of this neither local nor national authorities were keen to enforce Brown, which was shown by Governor Faubus. He decided to exploit white racism in this situation, in order to ensure re-election. In this way he stirred up the crowd and ordered the Arkansas National Guard to bar the school, sending a strong message of hatred to the black students. The images of harassment and violence towards the children by aggressive white adults, again, like in the case of Emmett Till acted as propaganda. This was a victory for the NAACP as it created a wide spread moderate opinion. However Faubus who closed all the schools in Little Rock in1959, preventing black or white from attending, in order to prevent integration, again showing that more than a court decision was needed in order to establish desegregation, again slowed progress down. As a result of the crowds and harassment of the children at Little Rock Eisenhower was forced to act. He had attempted to negotiate a settlement with Faubus, however the children were still not allowed to enter the school and the white violence in the streets became more aggressive, so Eisenhower sent in 10,000 troopers of the Arkansas National Guard. This was significant, as he had said that he could never envisage sending in federal troops to enforce federal court rulings. This was the only time in the 1950s that Eisenhower used his federal authority to intervene in the Brown decision. In this way the children were permitted to enter the school, signifying how the process could have been sped up, if he had acted sooner. However his actions were due to an, inescapable responsibility for enforcing the law, rather than a belief in integration. Again showing his lack of leadership and commitment to the cause, meaning that it was hard to put the brown decision into practice. In 1957 a Civil rights bill to ensure the black vote was proposed, however again this met criticism. Democratic senators worked to weaken the bill, as they thought it would damage national and party unity. Again Eisenhower showed little leadership, claiming that he did not really know what was in the bill and so did not fight to keep it intact. Along with the filibustering by Storm Thurmond, it resulted in a much weakened bill, not allowing blacks to exercise the right to vote as those who were prosecuted with obstruction would be tried in an all white jury and let off. This shows that from 1954-57 little progress was made, as although ideas are initially passed when put into practice it takes along time for them to be accepted, and so slow progress is made. To conclude from the evidence above it is clear that the building blocks of a strong civil rights campaign were begun between 1954-57, with the shocking legal decision of Brown Vs Topeka. However it is shown that although a decision was made in court, it does not mean that putting it into practice will be quick or easy. It shows that the more united the black community was and the harder that they pushed for change, the more stubborn the whites became, so making the changes impossible to implement without a fight. This case was not helped by the lack of leadership from Eisenhower, as his silence was interpreted as a lack of support for the cause, so discouraging other whites to back it. The lack of progress is shown by the fact that by 1964, a whole 10 years after the Brown decision, only 2/3 of the USAs black high school students attended de-segregated high schools. Therefore it is clear that between 1954-57 the achievement of civil rights progress was slow.