Business strategy analysis_ case Nucor corporation Essay

Business strategy analysis_ case Nucor corporation - Essay Example b) Analysis of Nucor’s challenges are exaggerated competition from global competitors, fluctuating steel cost, increased raw material cost, amplified energy cost and increasing costs to abide by the environmental convention (Nucor, 2013). d) Recommendation is provided to the company for dealing with their challenging issues. Nucor Corporation needs to practice pricing and revenue optimization which will prove to be successful technique for enabling the company to enhance profitability and competence (Nucor, 2013). e) Implementation of the plan can be possible by the contribution of the company’s readily available human resources, financial resources and time. The company has a strong organizational culture in combination with various technological innovations to support the growth plan. Nucor Corporation over the recent years has positioned itself as the fourth largest steel manufacturer by market capitalization. The chart below shows the increased revenue increase of the steel company over the two years (Whatley, 2013). The sales of the company has been rising over the several quarters, now having revenue increase of 15%. Due to the tremendous consolidation of the steel industry and monopolistic US steel market conditions, the company did not cut its prices much in order to respond to down market in the current years. Nucor Corporations net sales declined due to selling less steel and not lowering prices of steel. The company is making attempts to grow in the UK economic downturn and trying to build a long term value for their shareholders (Dzielinski, 2013). Nucor Corporation is also facing a tough competition from other leading foreign steel manufacturers which are offering steel products in highly competitive prices. Nucor Corporation has positioned itself as the global leader in environmental performance. According to the analysis of the case study the company has been able to make improvements in revenues and

Qualitative Research critique Essay Example | Topics and Well Written Essays - 2000 words

Qualitative Research critique - Essay Example In this, research becomes important in understanding safe and best practices (Barker, 2010). Additionally, research reveals information on other factors related to the profession and their effects. This essay seeks to analyze a research done by a group of nursing professionals entitled Men and Infant Feeding: Perceptions of Embarrassment, Sexuality, and Social Conduct in White Low-Income British Men. Just as is with any other profession, the use of current and relevant information in a practice is important. New and relevant information on any practice is only determined through research, researchers must therefore employ efficient and effective research methodology, data collection methods and data analysis tools capable of developing factual and relevant conclusions (Rees, 2011). The findings segment is normally the most important in any research; it is in this section that the researcher states all his new knowledge. Efficacy of a research is measured based on the techniques, trea tments, and technologies used in collecting and analyzing data. These three especially technology has kept changing through time; this therefore implies that knowledge held on a particular field need to change too. The Nursing and Midwifery Council recommend that practitioners pay close attention to the three in ensuring that knowledge in the field is fostered and developed though frequent researches. The frequency of research aids in developing a link with the society, this way the profession establishes a sense of currency with the trends in the society. Furthermore, researches should be as diverse as possible; this way they address all the possible factors affecting the profession. This essay through the use of a tool called the Critical Appraisal Skills Program (CASP), will analyze the use off the three: techniques, treatments and technology employed in the above stated research to evaluate the relevance and factuality of its finding therefore the efficacy of the claims it makes in its conclusion. This criticism extends to the research structure, terminology and the actual choice of words as used by the researchers in developing and communicating their research findings and actual ideas. These are important in any research as they aid understanding of the research report. Without following the accurate format and or use of correct terms, the report becomes confusing and may end up being of no relevance to the target audience (Brayford & Weston, 2008). Breastfeeding refers to the act of mothers feeding their young babies milk straight from their breasts. Health researches have established that breastfeeding has more health advantages to both the mother and the infant. A baby should be fed on the breast milk for a minimum of six months; such babies grow faster and develop stronger self-immunity. Effectively breast fed babies are always less likely to suffer from such diseases as respiratory infection, gastroenteritis, and urinary tract infection. In addition to faster growth, breast feeding is a means of communication between a mother and her baby. Effectively breast fed babies develop a unique attachment to their mothers making them understand their mothers behavior and intuitions. A mother on the other hand uses this as a means of monitoring the baby. Such elements of the infant as its mood and comfort are determined

The impact of gambling in America Essay Example for Free

The impact of gambling in America Essay The impact of gambling on the United States carries both positive and negative effects into our society and communities. Gambling is a leisure activity that usually provides excitement and fun for its players. Gambling also brings tremendous amounts of money into the economy and provides employment. American cities such as Las Vegas and Atlantic City thrive off of the gaming industry. Sure, gambling can be fun and exciting, but at what point does that fun activity develop into a pathological problem? I will contend that the harmful side- effects of gambling far outweigh any positive aspects. Pathological gambling can destroy families, careers, and lives. The reason why casinos are so successful is because their customers lose money while playing their games. Gambling is a broad subject in definition and is also known as â€Å"betting†. It is defined as â€Å"any behavior involving risking money or property on the outcome of a game, contest, or other event in which the outcome of that activity depends partially or totally upon chance or ones ability to do something† (wikipedia. org). In reality, someone could bet or gamble on just about anything. For example, I recently stumbled upon an online gambling site (www.bodog. com), which features a section where you can wager on who will win the reality shows on television. Gambling is most prevalent in card games, slot machines, dice games, sports, and horse racing. Gambling and the gaming industry in the United States have come a long way since their early beginnings. According to historical records and archeological evidence, gambling has existed throughout the ages of most civilizations. Anthropologists agree that data collected in the 20th century implies that gambling took place within a large portion of the greatest societies to have ever existed. For example, gambling artifacts have been recovered from ancient China, India, Egypt and Rome dating as far back as 2300 B. C. (GamblingPhd). Gambling has been around for centuries, but until recently, it has been restricted to back rooms, pool halls, hard to reach casinos, and Indian reservations. Geographic barriers, state regulation, and local ordinances effectively restricted the industrys growth (gsu. edu). Perhaps the most important reason why gambling is most prevalent today is because it is convenient. Large market companies, casinos, and the rise of the internet have paved the way for the industry to target potential gamers. I was on my way to Keeneland to watch the horse races this fall when I passed a sign that read â€Å"Drive-through betting†¦.. Gate 2†. While others may not have noticed, I was shocked to see this simple sign. No longer do you have to step out of your car to wager on horse races. This is just one example of the convenient way to gamble money. The unregulated and decentralized nature of the Internet creates the perfect environment for the growth of gambling. Anyone, anytime, anywhere with a computer, a connection, and a credit card is able to gamble online with thousands of websites dedicated solely to gaming (gsu. edu). Online sportsbooks and casinos get around federal laws by locating their company in a foreign nation, but still target the American consumers. Is online gambling legal for Americans? Often times, there is a â€Å"grey area† in the laws governing gaming products and online gambling. Internet gambling laws are also extremely difficult to enforce. The simple answer is there is an explicit law against online betting in only three states – Nevada, California, and Louisiana. Even so, no American citizen has ever been arrested for betting on the internet (about. com). These convenient methods are contributing to the growing popularity of gambling among Americans. Many people gamble socially without ever encountering a psychological problem. Often times they buy a lottery ticket, go to the racetrack, play a game of poker with some friends, or a night of bingo. When the activity is done, they go on to other non-gambling activities. But for some people, gambling becomes a big part of their lives. They are unable to stop, and believe in the â€Å"big win†. The problem gambler believes that the big win will solve all of their financial problems. They are always waiting for it, which causes them to lose more and more money. Rather than changing their gambling habits, they pin all of their hopes on the big win (aadac. com). A problem gambler will also boast about their wins often. Re-living their wins makes them feel more comfortable when they are actually losing. Legalized gambling brings both positive and negative economic effects to our society. It affects American communities in various ways. Gambling is helpful in our economy in two major ways. First, the gambling industry has provided an increase in employment opportunities. The NGIC reported that in 1996, the legalized gambling industry employed more than a half million people that earned more than $15 billion in salaries (gao. gov). Casinos have also created economic development by bringing in money from tourism, and increasing tax revenue and investment for communities. Charitable gambling benefits many needy causes in local communities. Native American tribes have also benefited enormously from legalized gambling. Casinos located on Indian reservations have allowed these tribes to grow financially and work their way into the U. S. economy. The negative economic effect that gambling has on communities is the increase in personal bankruptcy. In communities where casinos were introduced, there is usually a direct increase in personal bankruptcy. For example, in 1998 the bankruptcy rate in Atlantic City, New Jersey per 100,000 people was 1,019 while the rate of the state of New Jersey was only 555 (gao. gov). In 2003, there was a proposal to add casinos into the mix of Kentucky’s gambling options. Dr.John Kindt, a professor of commerce at the University of Illinois said that bringing casinos to Kentucky would be â€Å"bad economics and bad social policy†. â€Å"Studies show that within the 35-mile feeder market around a casino, gamblers will spend 10 percent less on food, 25 percent less on clothing and that 37 percent will dip into their savings to pay for gambling. For every three (video slot) machines, youll be losing two jobs from the feeder market economy, Kindt said. â€Å"This occurs because each machine can be expected to bring in $100,000 per year that will not be used elsewhere in the economy† (kybaptist.org). This is a serious issue that occurs when business owners do not think about how their actions will affect others in the future. Not only does gambling affect the economy of our communities, but it also has social effects. There are many negative consequences of gambling, while there are minimal positive outcomes. Beginning with the positive side, gambling is a recreation, a break from the everyday pressures of life and a relief from stress. People feel good about themselves when they do hit a jackpot or â€Å"win big†. Without gambling, certain sports such as horse racing and boxing would not be nearly as successful. Lastly, casinos do occasionally give back to the community and fund local and national charities. Socially, gambling has a greater affect on communities in a negative way. Three primary problems that occur are an increase in crime, suicides, and family problems. Individuals who suffer from problem or pathological gambling engage in destructive family behavior, such as domestic violence, divorce, and homelessness (gao. gov). Children of these individuals are often prone to suffer from abuse and neglect. In 1998, the NORC (National Opinion Research Center) estimated that the annual average cost to society for a problem gambler for job loss, unemployment and welfare benefits, poor physical and mental health, and gambling disorder treatments were approximately $1,200 dollars (gao. gov). The time that some of these gamblers spend at a casino is detrimental to their marriage, their children, and their job. Pathological gambling has been linked directly to increases in crimes committed. Whether it is robbery or insurance fraud, these types of gamblers are usually desperate for money to fuel their gambling needs. The counselor for the Department of Corrections in Wisconsin, Bill Schaff stated, In the most desperate phase of compulsive gambling, they will do anything to gamble. They start stealing money from their spouses; family . . . theyll get money anyhow, anywhere. Theyll do forgeries, embezzlements and thefts. Its mostly white-collar crime† (family. org). Lastly, the suicide rate among pathological gamblers is higher than for any other addictive disorder (gao. gov). With most businesses, their customers are assets and are catered to and cared for. In order for a business to succeed, they need their customers to return to them and remain loyal. Their customers are, in essence, cultivated. With casinos, their customers are not there for a service. They let the machines and games do the work, especially if they are the only casino in the area. What happens to their customers is not their concern. It is in the casinos benefit for the customers to become addicted. To casinos, they are resources that are strip-mined. There are four stages of gambling a person can go through while becoming a pathological gambler. The first stage is the reason why someone is gambling. They either gamble for the action or for escape. Gambling for the action begins when someone has success and thinks they are good at it. The excitement boosts their self-esteem and they continue in search of more money. Gambling for escape begins when someone is lonely or depressed. It becomes an outlet for them to escape from reality and their problems. The second stage is the gambler’s reaction to losing. Pathological gamblers â€Å"chase† after their losses. They find losing intolerable and focus on getting their money back. â€Å"Chasing† is easily avoided by setting a reasonable limit of money to spend in a day. The third is the desperation phase. In this stage, their losses become significant and their jobs and family come into jeopardy. The obsession with getting out of trouble overtakes the excitement of gambling. Crime and illegal activities occur in order to gain extra money. Serious bouts with depression and suicide attempts take place. They continue to escape from their lives by gambling, but find no relief. The final stage would be the hopeless stage where getting even financially would not be possible. The gambler knows they will lose and no longer cares. There are numerous organizations that focus on gambling behavior modification. They help pathological gamblers acknowledge their problem and overcome irrational thoughts and impulsive behavior. Gamblers Anonymous, Gam-Anon, and NCPG (National Council on Problem Gambling) all contribute to assisting problem gamblers and their families. In rehabilitation, gambling is treated the same as alcoholism, drug abuse, and sex addiction. Gambling is just one of the addictions that provide the users with a temporary, illusional escape from reality. The overall mission of all of these groups is to increase public awareness of pathological gambling, ensure the widespread availability of treatment for problem gamblers and their families, and to encourage research and programs for prevention and education (ncpgambling. org). Gambling addiction often leads to alcoholism and depression. It is important for family physicians to identify and treat this condition as a psychiatric disorder. The personal costs of gambling rarely gain public attention, but one prominent example is that of Pete Rose, who was denied induction into the Baseball Hall of Fame as a result of sports gambling. Rose, a pathological gambler, had bet thousands of dollars on baseball games while he was a player and manager of the Cincinnati Reds. He admitted that he did have some problems in his life, but said he did not bet on baseball. This is a prime example of a pathological gambler who repeatedly lied and ruined his life over gambling. One of the criteria for a gambling disorder is repetitive lying to conceal the extent of one’s involvement in gambling, much the same way an alcoholic, or drug addict tries to conceal their behaviors (about. com). Teen gambling is on the rise across the country, due to a couple of factors. One is the glitzy, celebrity-filled, high-stakes poker games that fill the cable TV channels. The popularity of Texas Hold’em poker was booming this year. The other is the cultural attitude that says gambling is fun, glamorous and possibly the source of vast wealth (about. com). Unfortunately, many parents view games as friendly, social events. They know where their kids are and assume their child wont develop a problem. Last Christmas, you could find poker sets targeting teens in almost every store. The fact is, that the risk of developing a gambling problem is the same for everyone, and possibly higher among teens. Teenagers are unable to make the same financial decisions as adults are, yet gambling companies continue to target the teenage consumers. The consequences of becoming a gambling addict are just as harsh for a teenager as they are for an adult. Once again, the internet and the ease of gambling are contributing to this problem. In an online environment of anonymous identity, the ease with which teenagers and children can access Internet gambling, coupled with their interest in gambling and poker, will only add to this worrisome trend. The sad part about the gambling industry and large market casinos is that they also target the elderly. Their propaganda reaches out and brings in the people who are unable to gamble responsibly. Casinos provide cheap buffets, coupons, drug discounts, and cater to elderly handicaps. They provide shuttles and buses from nursing homes and retirement centers. I have seen first hand how the senior citizens flock to slot machines and casinos. Often times they sit there for hours at a time with walkers, canes, wheelchairs, and even oxygen tanks. I have even heard stories of elderly gambling addicts committing suicide because they were so far in debt. The gambling industry is not a modern day Robin Hood that takes from the rich and gives to the poor. It takes from the elderly and the poor and gives it to the rich†, said Dr. Jimmy Porter, of the Mississippi Baptist Christian Action (kybaptist. org). Many of these people do not have the ability to control their gambling habits and do not realize how much money they are losing. An article in â€Å"The Call†, a Rhode Island newspaper is titled â€Å"A helping hand for seniors addicted to gambling†. It reads, â€Å"Sitting in her own urine, the elderly woman continued to play the game. Observers concluded she had some sort of bladder disorder, but the real problem was actually staring the woman in the face: the slot machine. Her gambling addiction had reached the point where she ignored everything even her own bodily functions simply so she could keep on playing† (The Call). Americans seem to clamor for seats at the blackjack table a lot more often than they do for seats in the ballpark. Now more people gamble at casinos than attend professional baseball games each year (aadac.com). This is a major statement about the changing values of American society. Located in twenty-three states, casinos gross more revenue than all sporting events, movies, plays, and concerts combined (aadac. com). In conclusion, the negative effects of gambling far outweigh the positive outcomes. The continued growth and convenience of the gambling industry raises concerns about a possible increase in the prevalence of problem and pathologic gambling. With the industry on the rise, the only solution to problem gambling will be to gamble responsibly. We need to ensure that all Americans have the information, skills and encouragement to reduce the risk that they develop a gambling problem, with special attention to those at increased risk. We must promote safe gambling practices and encourage people to set a limit. If we do not take action immediately, we are going to see the negative effects of gambling unveil themselves like never before. Pathological gambling is a serious psychological problem, and needs to be addressed just as drugs and alcohol are. Works Cited Baldwin, John. Ungar, Bernard. â€Å"Impact of Gambling. † April 2000.http://www. gao. gov/new. items/gg00078. pdf Bell, R. J. â€Å"Online Sports Gambling: The Law and You. † 10 Nov 2005. About. http://www. sportsgambling. about. com/od/legalfacts/a/betting_laws. htm Eadington, William. â€Å"Current Trends in Gambling. † 15 Sept. 2005. University of Nevada. http://www. unr. edu/gambling/eadington_papers Haynes, Jeff. â€Å"A Helping Hand for Citizens Addicted to Gambling. † 3 June 2003. The Woonsocket Call. http://www. zwire. com/site/news McMurry, Kevin. â€Å"Gambling on the Internet. † December 1999. http://gsulaw. gsu. edu/lawand/papers/fa99/mcmurry_thomas/. â€Å"Problem Gambling: The ABC’s. † 10 November 2005. AADAC. http://www. corp. aadac. com/gambling/the_basics_about_gambling. asp Reeves, Robert. â€Å"Expanded Gambling Bad For Economy. † Kentucky Baptist Convention. http://www. kybaptist. org/kbc/welcome. nsf/pages Reno, Ron. â€Å"Gambling and Crime. † 11 Dec 2003. CitizenLink. http://www. family. org/cforum/fosi/gambling/facts/a0029358. cfm â€Å"The History of Gambling. † 10 November 2005. Gambling Phd. http://www. gamblingphd. com/gambling-history. htm â€Å"The NCPG Mission. † 21 Nov 2005. National Council on Problem Gambling. http://www. ncpgambling. org/.

Expression and Purification of recombinant Green Fluorescent Protein

Expression and Purification of recombinant Green Fluorescent Protein ABSTRACT: The purpose of this experiment was to determine if a His-6 tagged recombinant form of Green Fluorescent Protein could be expressed in a pRSETA vector of E. Coli. This was determined through multiple procedures beginning with purifying the sample with Ni +2 agarose chromatography which showcased the relative fluorescent activity of the samples, which elution sample two (E2) had approximately 100,592.2 RFU/mg . The yield of total protein was found by use of a Bradford Assay and a standard curve. The purity of the GFP was determined by comparing the intensity of bands that appeared at around 31.4 kDa (the molecular weight of rGFP) to a molecular weight ladder on an SDS-PAGE gel. The Western Blot test, utilizing a nitrocellulous membrane, confirmed the expression of rGFP. The Western Blot confirmed that the correct bands were analyzed in the SDS-PAGE gel which E3 had an estimated purity of 0.4, indicating a yield of approximately 29.4 ug of rGFP for the third Elution (E3) after a total p rotein amount of 49 ug was extrapolated from the Bradford assay. INTRODUCTION: The Green Fluorescent Protein was first isolated from the Aequorea Victoria jellyfish and studied by Osamu Shimomura. In other organisms other than this specific jellyfish, there are fluorescent proteins with more color varieties than just green (Ward). GFP is able to fluoresce due to the formation of a chromophore in its center that resides in a stable beta barrel structure. The interactions between Ser64 and Gly67 on an alpha strand form a five member ring structure and its this structure that contains properties for fluorescence. When ultraviolet light is absorbed at 395nm, an emission wavelength of 510nm is seen as fluorescent green light. The excitation wavelength of 395nm excites the electrons within the protein and raises the energy of the protein. As the protein loses energy, it emits the energy at a wavelength of 510nm. Wild type GFP does not have immensely visible fluorescence, therefore a genetically modified form of GFP, GFPuv, was used in order to better observe fluoresc ent activity. For rGFP to be fully expressed, GFPuvs open reading frame was pRSETA vector, which was cut by a restriction enzyme at sites that complimented the start and stop codons of the His-6 tag sequence. A T7 promoter and Xpress Epitope were also genetically added into the vector with the purpose of simplifying purification. T7 polymerase minds to the T7 promoter and activates the production of GFP, while the Xpress Epitope allows for the binding of a primary antibody. The binding of a primary antibody allows for the binding of a secondary antibody which contains horseradish peroxide, producing bands on the nitrocellulose membrane. (2 pg 351) Histidine-6 tags primary purpose is to aid in the purification of rGFP proteins in affinity chromatography. Histidine has an affinity to bind with transition metal ions, and is why a Nickel+2 agarose chromatography was chosen. When a sample is passed through the Nickel+2 agarose column, the His-6 tags fused with rGFP protein will bind to the column and any proteins not of interest or containments will pass through the column. To elute the bound rGFP proteins from the column, elution buffer containing Imidazole is used and competes with the histidine residues for the binding sties to the Nickel+2 agarose column due to its higher affinity for them. Elutions from the column therefore contain rGFP. (2 pg 359) The purpose of this experiment was to see if a His-6 tagged recombinant form of GFP, from the E. coli strain BL21pLysS, could be expressed and then purified using Ni+2 agarose affinity chromatography technology. If this experiment is successful, an inexpensive and relatively simple method to measure gene expression and locate and track proteins may be at hand. MATERIALS AND METHODS: Bacterial Expression Grow two bacterial cultures: G, containing BL21, DE3, pLysS, and the plasmid pRSETA-GFPuv with the GFP sequence, and V, which is identical to G, but it does not have the GFP sequence. Incubate both cultures at about 37Â °C until OD600 equals 0.5. Transfer 1ml of each culture into separate centrifuge tubes and centrifuge to obtain a pellet. The supernatant from the tubes will be discarded and then label the tubes V0 and G0 and stored at -20Â °C. Induce the rest of the culture with IPTG and allow to grow for 3 hours. After the 3 hours, collect 1ml of each culture and centrifuge. Label the tubes V3 and G3 and were store at -20Â °C. Collect 15 ml of G and centrifuge, and label it as G3-15ml and store at -20Â °C. (1 pg 105) Preparation of Crude Extract After the slow freeze process, add 1 ml of breaking buffer (10mM Tris, pH 8.0; 150mM NaCl) to the frozen bacterial pellet G3-15ml, and pipette up and down until completely dissolved. Then transfer the contents to a centrifuge tube and vortex for 5 minutes and place in a 37Â °C water bath for 10 minutes. Afterwards, incubate in dry air at 37Â °C. Centrifuge the mixture at 4Â °C and transfer the supernatant into a clean centrifuge tube and take a small sample to label as GCE. The remaining supernatant will go through the Nickel+2 agarose column. (1 pg 110) Ni+2 NTA Chromatography Pack a 3ml plastic syringe with a small amount of glass wool to prepare the Ni+2 agarose column. After that, pipet 1 ml of breaking buffer into the syringe to remove any air bubbles. As the breaking buffer is going through the syringe, pour some breaking buffer into the leur-lock and then screw it onto the syringe. Add 1ml of 50% Ni+2 agarose into the column and then open the leur-lock to pack the agarose matrix in the colum. Add 5 ml of breaking buffer subsequently into the column to push the ethanol of the Ni+2 agarose through the column. Then close the leur-lock and apply the crude extract to the column and allow 5-10 minutes for the histidine tags on the rGFP to bind with the Ni+2 agarose beads. Open the leur-lock and collect the non-crude extract in a centrifuge tube labeled W1 for the first wash. Add 0.5ml aliquots of breaking buffer to the column and collect the washes in tubes W2-W10. Add 10 increments of 0.5ml of elution buffer and collect in tubes E1-E10. Then store your GC E, W1-10, and E1-10 samples at -20Â °C. (1 pg 111-2) Bradford Assay Analysis To create a Bradford assay, mix protein sample with water, and then add Bradford reagent dye. Vortex the mixture and then transfer it to the wells of a microtiter dish, so that the absorbance of your protein sample can be determined at 595nm using a spectrophotometer. To determine the amount of total protein present in the sample volume, you must create a Bradford standard curve using 0, 2.5, 5, 10, 15, and 20 ug of BSA and the absorbance values observed to determine a best-fit line. Perform the assay in singlicate using samples W1-W6 and E1-E6 to determine if the microplate data falls within the standard curve. Once you have determined what volume of sample to use, repeat the Bradford assays two more times for each sample. Use the standard curve and experimental absorbance values to extrapolate the total protein present in the volume of sample you use. (1 pg 124-6) SDS-PAGE/Coomassie Blue Analysis of rGFP Fractions Create a 12% Resolving Gel by mixing water, 30% Acrylamide, 10% APS, TEMED, and 4x resolving buffer [0.75M Tris pH8.8, 0.4%SDS]. Pour the resolving gel into a gel electrophoresis set up and overlay with some water. Allow the resolving gel to polymerize and proceed to making a 5% Stacking gel by mixing water, 30% Acrylamide, 10% APS, TEMED, and 4x stacking buffer. Pour the stacking gel on top of the resolving gel and immediately insert a comb to form the wells. Prepare your loading samples with 4x sample loading buffer and then vortex, boil, and centrifuge. Do this for the G0, G3, GCE, W2, W3, E2, and E3 samples (or fractions that contained the most rGFP fluorescence). (1pg 111-2) After the gel has solidified, transfer the apparatus into the electrophoresis tank and load the electrophoresis buffer into the tank. Then load your samples into lanes one through seven respectively along with a marking ladder to compare the distances the samples traveled after electrophoresis. Electrophorese at 200V for approximately 45 minutes and then stain your gel with Coomassie Blue dye. To remove the stain that has not been absorbed by proteins, the gel is soaked in dilute acid and methanol. Western Blot Develop an SDS-PAGE gel as previously described using your sample fractions and transfer the proteins onto nitrocellulose transfer membrane using two locking cassette lids with sponges and filter paper encompassing the gel and nitrocellulose. Remove the nitrocellulose and stain the membrane with Ponceau S stain, and allow to incubate for approximately 2 minutes. Once stained, wash the membrane with nanopure water until bands are visible on the membrane. Next, perform the blocking step by placing the membrane in a container containing 5% non-fat dry milk/TBS solution and incubate on a shaking platform. Pour out the blocking solution and add 0.05% Tween 20/TBS solution and then allow it to incubate on a shaking platform, and then pour out the solution. Repeat this wash step two more times. Add mouse IgG anti-Xpress epitope MAb and allow incubate on a shaking platform this is the primary probe step. Then repeat the wash step as mentioned previously three more times, and add Sheep IgG a nti-mouse IgG conjugated horse radish peroxidase polyclonal anti-serum solution (the secondary probe). Allow the membrane to incubate, and then perform the wash step twice. For the final wash, use only TBS, and then add TMB substrate solution and incubate until appropriate banding is shown. Stop the reaction with distilled water and scan or photograph the membrane as the results may fade with time. RESULTS: In the specific strain of E. coli, BL21pLysS, pRSET-GFP represses the lac promoter which is part of the operon needed for T7 polymerase activity. T7 polymerase binds to the T7 promotes of pRSET-GFP and activates the His-6 tag that expresses the fluorescence of GFP. Even though some GFP is produced this way, it is experimentally not enough, so IPTG is added to repress the repressor so an increase of GFP production can occur. V0, V3, G0, and G3 were all bacterial cultures of E. coli with specific distinctions between all of them. V0 and G0 were collected from bacterial cultures that were not yet induced, but G0 contained the GFP sequence while V0 did not. V3 and G3 were the V0 and G0 samples after induction had occurred for 3 hours, and again G0 contained the GFP sequence while V0 did not. W1-W10 were washes collected from the Ni+2 agarose column using the breaking buffer whereas E1-E10 were washes collected using elution buffer. Figure 2. SDS-PAGE/Coomassie Blue stained gel analysis of rGFP SDS-PAGE gel, which was used to analyze our purification of rGFP from a crude extract, and the corresponding molecular weight. This gel was made with a 12% resolving gel and a 5% stacking gel. Wildtype rGFP has a molecular weight for 27kDa. The ladder has kDa rungs of 97.4, 66.2, 45.0, 31, 21.5, and 14.4. The band for rGFP has been indicated on the figure. The different samples were taken during our purification. The highest protein containing washes and elutions were used in the SDS-PAGE gel. G0 represents a sample containing rGFP prior to induction. G3 represents a sample containing rGFP after 3 hours of induction. GCE represents the GFP crude extract after the slow-freeze/quick-thaw process. Figure 3. Western Blot of rGFP We used the Western Blot to detect, and more importantly, quantify proteins that react with antibodies. The Ponceau S stain was performed to highlight the existence of our proteins successfully being transferred from the SDS-PAGE gel. We then performed a blocking step with 5% non-fat dry milk/TBS solution followed by washes with 0.05% Tween20/TBS. The probe first used was mouse IgG anti-Xpress epitope MAb solution, which we followed with washes of Tween20/TBS solution. The second probe used was sheep IgG anti-mouse IgG conjugated horseradish peroxidase polyclonal anti-serum solution, which we followed with two washes of Tween 20/TBS solution with the third wash being pure TBS. Then, TMB solution was added to develop the nitrocellulose membrane and this reaction was stopped by pouring distilled water over the membrane. Above are the 8 lanes, G0, G3, GCE, W2, W3, E3, E4, and a known molecular weight ladder, respectively. CONCLUSION/DISCUSSION: A great amount of research has been put into the field of monitoring gene expression and there are currently many research laboratories that use labeled antibodies and other various means to do so. However, this new recombination technology, a vector can be created to provide a simpler and less expensive approach that can even be performed in vivo. The purpose of this experiment was fulfilled as His-6 tagged rGFP from a particular E. coli strain was successfully expressed, purified, qualified, and quantified by means of Ni+2 agarose chromatography. GFP was initially expressed as a histidine tagged protein that was inserted in E. coli and subsequently a crude extract of this protein was isolated. Throughout the experiment, we were able to inspect and measure the fluorescing activity of our elutions with the use of handheld UV lights. GFP was successfully expressed because of the fluorescent activity that was emitted when shown under UV light. This rGFP was purified using a Ni+2 agarose column which allowed for the binding of the His-6 tagged rGFP to the Ni+2 agarose beads in the column, and subsequently let all other proteins not of interest and containments to be collected in washes. Then, rGFP proteins were eluted by passing elution buffer containing Imidazole through the column because Imidazole has a higher affinity for the Ni+2 agarose beads than does Histidine. Therefore, one could expect that most if not all of the rGFP would be found in the elution washes and would present higher fluorescing activity than the washes with breaking buffer. Figure 1 clearly supports this argument. From referencing Figure 1, the most rGFP was found in E2 because it exhibited the highest fluorescent activity in RFUs. Using a standard curve based off of known quantities of mass of a certain protein, one is able to extrapolate the amount of protein present in ones sample. This can be used to estimate the amount of rGFP activity. For instance, E2s specific activity was found to be approximately 100,592.2 RFU/mg. W1-W6 had the least amount of fluorescing activity because the washes were done prior to the addition of Imidazole through the column. This is expected as Imidazole would flush out rGFP in the elutions, which would produce the highest GFP fluorescing activity. Percent purity of the rGFP samples were approximated using the SDS-PAGE gel with Coomassie blue staining to allow visible bands to appear which were used for comparisons. The molecular mass from each individual sample was determined by comparing the bands with the bands of the known weight ladder. The percent purity of each individual band was determined by comparing the bands against each other. The molecular weight of wild type rGFP is 27 kDa, and the molecular weight of rGFP for mutant rGFP used for this experiment was approximately 31.4 kDa with the 40 extra amino acids that were added taken into account. For example, E3 percentage of purity was found to be .4. The amount of protein yielded was 29.4 ug of rGFP for the third Elution fraction (E3) after a total protein amount of 49 ug The Western Blotting technique detected and quantified proteins that reacted with a specific antibody. In our case, it verified rGFP was expressed and whether or not it fell in the range of the expected molecular weight of 31.51 kDa. Lanes GCE, G0, G3, and E3 all contained a bright band around 31.4 kDa, indicating that rGFP was existent. This result corresponds to the expected of E3 having the one of the highest rGFP activity. The fact I had used E3 for the SDS-PAGE and Western Blotting skewed my results, but E2 and E3 had the highest activity for me. Even with my error, GFP was effectively expressed and purified and its finding is important to science because it can be used to track cancer causing cells one day to help figure out where problem spots begin and can help eliminate the issue before it escalates indicating that fluorescent proteins might eventually be clinically useful in cancer patients (Hoffman). This can occur since GFP can successfully be expressed in other organisms as this experiment through the use of E.coli, humans cells can certainly be tagged and label the tumour before treatment and then monitor for fluorescence after treatment to identify possible recurrence or metastases (Hoffman). The same can be said with heart disease and other health related problems. The future of GFP remains limitless when scientists can unlock its full medical potential. REFERENCE/BIBLIOGRAPHY (1) Rippel, Scott. BIOL 3380 Fall 2010: Biochemistry Lab Manual. The University of Texas at Dallas. Richardson, TX. (2) Rippel, Scott. BIOL 3380 Fall 2010: Biochemistry Lab Lecture Notes. The University of Texas at Dallas. Richardson, TX. (3) Ward, William. History of GFP and GFP Antibodies. Brighter Ideas. 2009. 26 Oct, 2010. http://www.brighterideasinc.com/proteins-antibodies/history-of-gfp-and-gfp-antibodies/ (4) Ninfa, Alexander J. and David P. Ballou (1998). Fundamental Laboratory Approaches for Biochemistry and Biotechnology. (5) Hoffman, Robert M. Uses of Fluorescent Proteins to Visualize Cancer In Vivo: Can Fluorescent Proteins be Used in Humans? 2005. 28 Oct. 2010. http://www.medscape.com/viewarticle/513975_6

The Invisible Poor :: essays research papers

I believe that the wealthy Americans have moved farther away from the poor then in past generations. James Fallows in †The Invisible Poor† clearly shows how the new technology millionaires awareness of the poor has diminished greatly. I believe that this is due to several reasons the most important being the young age in which wealth and success are reached. The technology millionaires are much younger then previous generations of millionaires and thus are removed much earlier one. They find success very easy and can not see why others can’t obtain the same wealth as they have. They also limit their circle of friends to those with similar interests or backgrounds. They have limited time and only spend what free time they have (beyond family and work obligations) with those who they are comfortable with. This comfort is generated by their common threads such as education, similar employment or social activities. When the rich stay within this circle they no longer are exposed to the poor and thus they forget that they exist. When they are exposed to the poorer people it makes them uncomfortable (like when the cleaning lady was around) instead of dealing with it or helping they choose to avoid the situation. Figuring if you avoid it then you’re not admitting that it exists, thus not having to deal with them. I believe that since the article was written times have changed somewhat with the collapse of the stock market in a sense. Many people losing their jobs in the high tech world may bring some of them back to reality and acknowledge those less fortunate. They are now looking for jobs and finding that their skills were limited to the high tech industry. Many are now taking lower paying jobs to get by. They have discovered that the wealth of stock options are now worth allot less. Making many of them near poverty themselves.

Native Americans

For many years Native Americans life has been a struggle. The government at the time was trying to encourage them into the white world and ultimately lose their cultural beliefs but Indians themselves at the time fought bitterly against the policies by the US government and also for their tribal lands. Their major concern was that if they chose to live away from tribal land they experienced discrimination and if they lived in tribal land the US constitution did not apply to them. In 1961 tribes gathered for a meeting in Chicago where they issued declaration of Indian purpose that stressed responsibility of preserving their native land and the right to choose their own way of life. Due to the activism the congress passed the Indian Civil Rights Act of 1968 which granted Native Americans full access to the United States Bill of Rights and protection for their tribal lands. This means that the Native Americans can govern themselves in sovereignty. Even though civil right gave tribes legal rights and protection there were conflicts that arose between respecting Native American sovereignty and practicing the United States bill of rights. Robert, Ericson and D. Rebecca Snow, â€Å"The Indian Battle for Self-Determination†, 1970 this additional source was an article written by two scholars who explained the battle for self-determination for the Indians. The article provides new information about the controversy in American courtrooms and legal halls. The article stated, â€Å"Federal courts have traditionally viewed the tribe as a dependent or â€Å"tributary† nation possessed of limited elements of sovereignty and requiring federal protection;' the Congress has alternately viewed the tribe as a substantially independent political unit or as an anachronism which must be phased out consequent to a policy of Indian assimilation† (Erickson and Snow). This quote shows us that the tribal sovereignty has often been conflicted. Another important information I learned from the article was what a tribe cannot do when it comes to certain situations. The article claimed, â€Å"Moreover, a tribal Indian cannot claim protection from illegal search and seizure, or from interference with freedom of speech, press, or assembly. In addition, a tribe can impose a tax, or revoke tribal membership rights without complying with due process requirements† (Erickson and Snow). This quote explains the limitations of being a tribal Indian. This was an important information to learn because it showed the struggles of many Indians back then. Lastly, this quote explains one of the positive aspect of the civil rights act of the government. The quote states, â€Å"Perhaps the most encouraging aspect of the proposals is that they represent an attempt to respond to Indian grievances. This is a refreshing departure from past policy. Too often, Indian preference has been ignored† (Erickson and Snow). All of the examples provide new outside information about the Indian civil right Act of 1968. Finally, this source could be consider trustworthy and biased because even though it is written by a scholar but it is not like a newspaper from during that time of the event. I think that both of the authors agreed on the Indian civil right act but they states that there were conflicts and controversy in American courtrooms and legal halls. Both of the scholars liked how the government took an attempt to respond to the Indians because they often get ignored. The article was well balanced when providing information because not only it talked about the rights act but it also provide information about the government and how the Indian civil right act movement started. I think this was a good source to use because it gave me new insight about Indians and I learned more information about the civil right Act Itself.

Lord of the Flies: Intelligence and Good vs. Evil Essay

Good versus evil is a common concept used often in storytelling, writing, plays, movies, etc. the basic story line is commonly used and developed to mold different ideals, meanings, and lessons into different types of works. William Golding’s novel the Lord of the Flies falls into this category of works with the good versus evil story line. Boiling the novel down to its most basic state it is a story of a group of boys. They all start out in a state of innocence, then as they adjust to their new surroundings after being stranded on a deserted island with no adults; they chose whether or not they turn from their innocence. When the boys turn from their innocence they go from being good to evil, or as the interpretation of this novel is commonly perceived the boys go from being civilized boys to savages. It is in this sense that civilized and good can be used interchangeably for this interpretation, and the same for evil and savagery. Golding puts an interesting twist to this basic plot right in the beginning of the story. Golding takes the story from just a simple tale of good versus evil to good versus evil with competing ideas of intelligence. Right in the beginning of the novel we see these ideas of intelligence take form. As the story builds the differences in the types of intelligence grows and becomes more distinct. From there the competition of good versus evil begins. After their plane crashes the boys who were on the plane to escape the warfare in England are scattered on the island. Ralph, the first boy we are introduced to meets another boy named Piggy. â€Å"It’s a shell! I seen one like that before. On someone’s back wall. A conch he called it. He used to blow it and then his mum would come. It’s ever so valuable-â€Å"(15). With Piggy’s help Ralph uses the conch they found to call out to the other boys on the island. This is the first hint at the two types of intelligence. Piggy is already exhibiting signs of natural intelligence. This type of intelligence is developed based on the sensory analysis of the surroundings. This intelligence is more advanced and enables Piggy to think in more civilized, advanced ways. He is immediately made fun of for his appearance and as the story progresses is mocked as a know-it-all. No one listens to Piggy, even though the conch and the meeting were his ideas no one realizes this, nor do they care. Once all the boys are gathered together there is a vote on who should be chief. A boy named Jack is introduced as the leader of the choir boys and he wants to be chief, but when put to a vote Ralph is elected. Ralph does give the choir to Jack and asks what they would like to be. Jack tells Ralph that he and his choir shall be the hunters. Ralph depicts more social intelligence. Ralph knows how to work a crowd, how to lead a group, and how to gain respect. â€Å"Everybody must stay round here and wait and not go away. Three of us- if we take more we’d get all mixed, and lose each other- three of us will go on an expedition and find out† (23-24). This act showed that Ralph was able to get the attention of the boys at any time and that the boys would actually listen to him. His ability to be able to accomplish this as quickly as he did really shows his true social intelligence. â€Å"If a ship comes near the island they may not notice us. So we must make smoke on top of the mountain. We must make a fire† (38). Ralph does show some natural intelligence as well, but not nearly as much as Piggy exhibits. Jack also shows a form of social intelligence. He is able to keep command over his choir and they listen to him no matter what. This shows that in the beginning of the story even though the boys are all different, have different types of intelligence, and have mixed feelings about the situation they are still united together as a whole. â€Å"All day I’ve been working with Simon. No one else. They’re off bathing, or eating, or playing† (50). The boys begin to slack and begin to realize that there aren’t consequences or punishments for the wrongs they do. Jack and his group of hunters become obsessed with the idea of killing a pig, and are the only ones exempt from helping build shelters, though the other boys don’t really care enough about their orders to help build or gather food. It’s at this point in the novel where there are serious signs of cracks and issues with the order system the boys have put in place since being on the island. Jack is beginning to slip into a more savage state, and is using his social intelligence to bring other boys down with him. The improvised form of society that the boys have created is already starting to weaken and fall. â€Å"Kill the pig. Cut her throat. Spill her blood† (69). â€Å"Look! We’ve killed a pig-we stole up on them-we got into a circle-â€Å"(69). It is at this point in the novel where the group of hunters, the boys who killed the pig, have turned from innocence. The kill they had obsessed over finally happened and they were proud of themselves for killing. The act of killing a living creature, something they never had done before, was the true turning point for them. There was no turning back from it; the hunters even painted their faces, this sense of wearing a mask, pretending to be someone else enabled them to kill. â€Å"‘But they’ll be painted! You know how it is. ’ Eric says. The others nodded. They understood only too well the liberation into savagery that the concealing paint brought. ‘Well, we won’t be painted,’ said Ralph, ‘because we aren’t savages’† (Golding 172). Ralph is starting to understand that Jack and the group of boys who follow him are starting to turn to a more evil state. Ralph still tries to use his social intelligence and command over his boys so they don’t go over to Jack’s side. Within Jack’s group the obsession with hunting caused the boys to let the fire go out, and subsequently lose a chance at rescue. The hunters didn’t really seem to care about the fire; they were still overly hyped up about their kill, swearing that they would hunt again and bring back even more meat. As the boys become more and more content with letting go with the rules they get closer and closer to losing their sense of civilization completely. â€Å"‘If I blow the conch and they don’t come back; then we’ve had it. We shan’t keep the fire going. We’ll be like animals. We’ll never be rescued. ’â€Å" (Golding 92). As the boys start to fulfill their own wishes of hunting and playing they get sick of listening to Ralph and being controlled by the conch. They don’t want to do work; they just want to have fun. Jack rebels against Ralph and makes his own â€Å"tribe,† where he could be the leader and he would no longer be controlled by the conch. Evil starts to arise rapidly; the majority of the older boys side with Jack and abandon Ralph’s authority. After that Piggy, Sam and Eric are the older boys who remain with Ralph. As the night wears on, some of the â€Å"littleuns† sneak off to join Jack. This is truly the end of their makeshift society, it has completely failed. After the split of the group it appeared as though things could settle down, but Ralph and his boys find that it is nearly impossible to keep the rescue fire going. They need to ask the others for help but Jack’s boys are too afraid to help them. Jack had taken to using torturous and intimidation methods to keep control over his group. He wears a mask, like he did when he killed the first pig; this seems to allow Jack to give in completely to his â€Å"evil† savage side. It was while celebrating their savageness and reenacting the kill that they got carried away and got themselves overly worked up. â€Å"The beast struggled forward, broke the ring and fell over the steep edge of the rock to the sand by the water. At once the crowd surged after it poured down the rock, leapt on to the beast, screamed struck, bit, tore. There were no words, and no movements but the tearing of teeth and claws† (Golding 153). The wickedness in all of the boys deludes them into mistaking Simon, the only truly â€Å"good† pure boy, as the beastie. Simon was considered to be the â€Å"pure† boy, the epitome of civilization and innocence. Jack and the other boys had completely given themselves over to their evil sides, giving in to their natural instincts and savagery. Social Intelligence and natural intelligence are both qualities that help a person to be successful in life. However, overall, society puts greater worth in social intelligence. The abilities to work with others, lead and motivate others, and inspire others make a person successful in life. Just like good and evil, social and natural intelligence go hand in hand. One is not greater than the other, but like people believe good is better, people put more stock in social intelligence. In Lord of the Flies the boys put their trust in Ralph, but as time goes on, and rules begin to be broken, the less control Ralph seems to have. Social intelligence will only go so far, it can’t be natural instincts. Natural instincts are hidden behind the rules and expectations of society. When they begin to dwindle and those instincts become more prominent, that natural intelligence becomes important, it can be the intelligence or lack thereof that can make or break a situation. Without one, evil or good, natural intelligence or social intelligence, the other is not really known. Without evil how would we know what good is? If everyone was socially intelligent how would we know what natural intelligence is? The answer is we wouldn’t have a way of knowing. A person cannot know one without knowing of the other. Intelligence and good and evil all work together, as seen in Golding’s novel. Jack possessed the same kind of social intelligence as Ralph, but when he gave in to evil his social intelligence the power it gave him over the other boys allowed Jack to change the atmosphere of the island and turn the boys away from goodness and civilization. Piggy had natural intelligence but because he lacked any real social intelligence he was shunned, mocked, and in the end killed for this. He came off as a know-it-all and a whiner; this annoyed the other boys and eventually led to his demise. The boys didn’t put any stock in natural intelligence; they put all their stock in social intelligence and natural instinct. Ralph, with Piggy’s help, was able to remain civilized and good. Though he lost his hold on the boys he still had his social intelligence, and even some natural intelligence of his own to rely on. When Ralph was in charge his positive outlook affected the boys as well, they were positive because he was positive. The struggle between good and evil does not just affect the person who’s battling it within themselves, it also affects those around them, and subsequently can change them too. ?