The 2001 MTV VMAs-An Overview essays

The 2001 MTV VMAs-An Overview articles 2001 has been an extraordinary year for the music and diversion channel, MTV. New periods of The Real Word and Road Rules, different shows and specials, and the Movie Awards have all been huge features of this current year up until now. The channel likewise praised its twentieth Anniversary this past August. Be that as it may, seemingly, the greatest occasion of them everything is the Video Music Awards. Known for its propensity to stretch the limits with regards to what can and cant be appeared on satellite TV, the VMAs are more about the show than the honors. This year was no exemption. Live from the Metropolitan Opera House, the show opened with have Jamie Foxx playing out an exceptionally fitting drama interpretation of a few of this previous years most famous melodies. At that point, he immediately changed gears into a monolog, offending a few big names that were available at the honors, as opposed to prior reports that he wouldnt do as such. Fortunately, Will Ferrell of Saturday Night Live notoriety ran onto the phase in Foxxs act, ascending a prop in front of an audience while yelling, Im seething! This was a reverence to a comparable demonstration performed by the bassist of the band Rage Against the Machine from a years ago VMAs, and constrained MTV to slice to a business break. In this manner started a consistent stream of exhibitions and grants. A fascinating subject to the VMAs this year was the wilderness. Before the chosen people for each grant were named, little nature narrative clasps were appeared, in any case, in evident MTV design, a large portion of them were somewhat provocative. Britney Spears lip-synchronizing execution likewise highlighted a zoo-loaded with confined creatures behind her, also a snake around her neck. Another snapshot of the VMAs that can't be neglected is the tribute to Aaliyah. A few specialists including Janet Jackson and Missy Elliot said a couple of words. Aaliyahs sibling additionally delivered a somewhat moving discourse. The second wouldve been great, had Janet Jackson not read t ... <!

Ethics in Retail Industry free essay sample

The investigation additionally includes how associations guarantee best moral practices and whether a socially dependable retailer increases upper hand and productivity through its moral practices. 25 December 2012 00:27 Methodology: Studying on previously mentioned issues can be begun from prevailing upon how morals and social duty affected the hierarchical procedures as of late. A short investigation of the retail associations, known as ‘ethical’, uncovers how the associations are pressurized to make such morally maintainable picture for its clients. Examining different models, the variables, impacting the moral acts of an association, can be identified with discover the stuff to be characterized as a socially dependable retailer. At long last the between relationship of gainfulness and moral practices can be set up to close the exhibition of retailing industry as far as morals and social duty. The methodologies on moral acts of certain association can be gotten to from the cases concentrates on these retail enterprises. The powerful factors can be recognized from the regular patterns in these methodologies and a decisive story through client observations can be drawn by direct customer contact and individual meetings. As a foundation of any association and individual while cooperating with different partners, inside a retailing industry certain measures are followed to manage the dynamic minutes. The extent of the article incorporates investigation of these practices according to moral perspective, taking models from goliath retail chains and furthermore investigation of moral view of the business power in retailing industry. The examination additionally incorporates how associations guarantee best moral practices and whether a socially dependable retailer increases upper hand and productivity through its moral practices. *Methodology:* [Quoted content hidden] 2 of 2 31/01/2013 10:37 PM Executive Summary: As a foundation of any association and individual while communicating with different partners, inside a retailing industry certain principles are followed to manage the dynamic minutes. This article incorporates investigation of these practices according to moral perspective, taking models from mammoth retail chains and furthermore investigation of moral impression of the business power in retailing industry. The investigation additionally includes how associations guarantee best moral practices and whether a socially mindful retailer increases upper hand and productivity through its moral practices. Different issues that may emerge from morally grieved circumstance where retail sales reps may wind up in moral predicament are talked about in various casing of references. Regularly normal untrustworthy practices in the retail business, for example, abuse of laborers, overemphasis on beneficial items, dishonestly fixing costs on items, exploitative issues of limited time crusade and their negative consequences for buyer mentality are talked about in detail. Subsequently untrustworthy practices in green retailing, the most recent buzz of the retail business in India are likewise talked about with models from universal retail chains. Through significant investigations it has been discovered that for retailers there are two sorts of moral code by and by: express and verifiable. Unequivocal code of moral practices indicates composed standards that retailers follow to work under. Certain code is characterized as the arrangement of unwritten however surely knew rules/norms of good obligations. In retailing condition Sales individuals interface with people from outside the store, clients and just as individuals from inside their association. The people with whom they cooperate may have certain dissimilar needs and issues that sales reps may fulfill to determine. Since these people need the sales reps to help cultivate their own need, fulfillment or issue, the requests conveyed to the salesmen by them are probably going to be different and regularly inconsistent. Therefore retail deals work force regularly face moral issues when conflicted between short run pressures from the executives I. e. to accomplish deals share and since quite a while ago run objectives of accomplishing client certainty and altruism. Furthermore the condition that the retail faculty works in is favorable for the improvement of moral issues due to the assortment of assignment they performs 3 hich can put them in awkward circumstances that may be conveniently tended to utilizing faulty conduct I. e. taking care of client return or trade. Further examinations have demonstrated that ordinarily retail deals work force get minimal formalized deals preparing and subsequently they have not been successfully taught about how the organization might want them to act in these mora lly upsetting circumstances. Administrators in retail industry ought to be worried about these circumstances looked by retail salesmen as failure to deal with such circumstance brings about bringing down occupation execution, disappointing client and so forth. Kinds of Ethical Problems in Retail Sector: There are various sorts of circumstances where moral issues can be happened in retail industry. From the differing individuals that retail sales reps connect including clients, friend and the executives anybody can imperil the moral standards of the circumstance. Utilizing these three gatherings of individuals as edge of reference moral issues can be sorted in three expansive territories; client, companions and business related circumstances. Client related circumstances may incorporate those circumstances that involve association with client and have direct effect upon clients. For instance neglecting to help clients or deliberately giving them off base change. Friend related circumstances include peer from non-work gatherings and individual representatives too. For instance offering or being compelled to give a companion or worker a markdown. Business related circumstances incorporate conditions that might be encouraged by organization working methodology, rehearses that are cultivated by workplace. For instance neglecting to acquire a check approval, selling an item as selective when it isn't could be remembered for exploitative practices. Basic untrustworthy practices in Retail Marketing.

Small Business Advise and Discussion Essay Example | Topics and Well Written Essays - 500 words

Private company Advise and Discussion - Essay Example These preparations incorporates the accompanying: 1. Legitimate Market Study: Analyzing the allure of the business, request and gracefully examination, client, contender investigation, SWOT examination of the organization, capital planning and money related getting ready for the business will help the entrepreneur to see all the little and enormous issues associated with the specific business. The objective market ought to be recognized and divided. The entrepreneurs face issues in raising the capital because of different reasons. These reasons can be chance, validity of the entrepreneur, advertise estimation of the tasks, anticipated development of the undertaking or the business, client base, business know-how of the proprietors, capabilities, experience and reinforcement to help any sort of monetary emergency. There are different elective wellsprings of account accessible for the little speculators. These are Boot Strap, Angel, Initial Public contribution and private placement.1 In the Boot Strap subsidizing the assets are raised with family, companions and others. On account of Angel the venture is finished by well off people. Private arrangements are done through banks and different ventures organizations. Organizations likewise access to the market through IPO to raise the capital. Boot Strap is a financing choice accessible for all sort of business. A large portion of the organizations fire up with the ventures this way. These sort of ventures which are finished with the assistance of different close circle individuals like family, companions, associates and at times great clients help entrepreneurs for their underlying phases of advancements. Entrepreneurs can successfully use these ventures. The outcomes delivered like benefit, expanded market esteem, client base and request of the items can pull in speculators for the further ventures. These can be new speculators, banks and other potential financial specialists. This can help the entrepreneurs to bring the assets up in the extension and development periods of the

The Forbidden Truth About The Road Mccarthy Essay Topics Exposed by an Old Pro

The Forbidden Truth About The Road Mccarthy Essay Topics Exposed by an Old Pro Finally nice and democratic governments ought to be instituted (Wannebo 97). It means finding different sources of food that might be more abundant. There are those out there. Also, you might wish to have a look at the how-to sites given below. A great deal of writers that are deemed good I consider strange'', he explained. Citations and extracts from assorted sources have to be formatted properly. If you get a compelling reason to find the original, talk to a reference librarian. Both subsequent sentences You aren't going to face the reality. It tells the reader what to anticipate from the remainder of the paper, how you are going to tackle the matter, and directly answers any questions asked of you. This book isn't poetry, however it's likewise not pure narrative. You will not discover any quotation marks within this book. The story ends on an excellent note. The Benefits of the Road Mccarthy Essay Topics Another intriguing moment in this portion of the book is whenever the man claims they must not stay there, although it looks like paradise. There's popular apocalypse, and then there's its antithesis. It's a post-apocalyptic tale told in an extremely minimalist style. Another illustration of symbolism is the use of one gun to signify hope and salvation. Road Mccarthy Essay Topics Ideas Finally, the man dies. He knows that he is sick and knows that he's dying. When he says that he will protect the boy at any cost, it is not an understatement. Another man appears after a number of days. At length, the primary goal of the guy and the boy throughout the full novel is to get to the coast. He decides that his sole purpose in life is to protect the boy from anything that poses a threat to him. It's also telling about the essence of the connection between a guy and a woman and their roles in a family. The guy and the boy fall within this category. The love because of his boy permits him to swear in the male child and starts to realize that the male child is capable of doing the most suitable determinations. Although the male child would like to compose a message in the sand to the fantastic cats the male parent finds it tough to continue being positive. It is hard to erase the image of a youngster and burnt flesh, especially given the simple fact they do not own a father to care for them. In clip the male parent arrives to observe how much his boy has matured and is ready to do the perfect determinations. Unfortunately, the website is too exposed, and they're ma de to move on after a couple of days. All images can be seen at a big size when you're in any reading room at the Library of Congress. No, the product isn't digitized. Yes, it is digitized. Maybe you'll have some luck. My occupation is to look after you. It doesn't really will need to get said that there isn't much hope to be found in a post-apocalyptic setting. The author endeavors to demonstrate the evolution of a human character during the principal protagonists. The Road Mccarthy Essay Topics and the Road Mccarthy Essay Topics - The Perfect Combination The setting is a somewhat depressing scenario in general. The key characters start to starve till they discover another hiding place containing supplies. You should have all pieces of the test under control before you demonstrate your skills in their entire entirety. It won't be any different than every other blood test you've received. What Everybody Dislikes About the Road Mccarthy Essay Topics and Why Ultimately, the book speaks to every one of us. The notion that the days are becoming worse and more morbid than each one prior is extremely clear and it's continuously stressed throughout the book. The guy and the boy, who also stay unnamed throughout the whole novel, travel during the rough terrain of the southeastern United States of america. So, it is going to expand, McCarthy states. the Road Mccarthy Essay Topics: the Ultimate Convenience! The middle of the world is sickened. The author appears to be pointing to the simple fact that with such cruelty, human are likely disappear from the face of the planet. The hope there are others like them that want to rebuild society sustains them. He portrays a lovely soft trout much enjoy the stunning soft world the way it once was before the catastrophe. the Road Mccarthy Essay Topics - Dead or Alive? Mentioning to how it's close impossible to keep up your religion in such difficult times. Folks are in a state of disillusion and it's even tough for one to imagine that things could possibly get much better. By making a place so desolate and hopeless, McCarthy managed to create a need to set up a reason to call home. The undesirable ones appear to take part in acts that demean and even eliminate the ones that are weaker than them.

Race And Ethnicity Cape Verdean American Immigrants Essay

As I read Marilyn Halter’s book, Between Race and Ethnicity: Cape Verdean American Immigrants 1860-1965, I was able to develop a clear perspective of the Cape Verdean’s American voyage as well as their social and economic triumph. Prior to reading this book, I had no knowledge of the Cape Verdean people, unless they are very similar to the â€Å"Brazilians†. Marilyn intentions for her book was to address the social construction of Cape Verdean racial and ethnic identity and how the trials they experience while margining into American society. Cape Verdeans, a mix raced people group of Portuguese and African decent, struggled to attain and maintain their social identity in America, all while enduring isolation and ridicule from both Whites and Black in the new world. After reading this Halters book and her narrative depiction of the Cape Verdeans experience migrating to America. Just as many American immigrants during early 19th and 20th century, they were in sear ch of an opportunity toward social mobility and sustainability while departing from the racial boundaries in their country. Though as soon as they arrived, the American society stressed the Cape Verdeans to choose to identified as white or Black? Little to their knowledge, their journey in search of prosperity immediately immersed them into the vulgar, racial divide between black and white Americans. The Cape Verdeans were a people group that migrated from the Cape Verdes Islands, not too far off the coast West Africa.Show MoreRelatedImportance Of Race Ethnicity : An Exploration Of Asian, Black, Latino, And Multiracial Adolescent Identity Essay1047 Words   |  5 PagesImportance of race-ethnicity: An exploration of Asian, Black, Latino, and Multiracial adolescent identity Article source- Charmaraman, L., Grossman, J. M. (2010, April). Importance of race-ethnicity: An exploration of Asian, Black, Latino, and Multiracial adolescent identity. Cultural Diversity and Ethnic Minority Psychology, 16(2), 144-151. doi: 10.1037/a0018668 (a) Contextual information about the purpose/intention of this study: Throughout the history of United States, race has been one of

Accommodating Employees With Disabilities At The Workplace

ACCOMMODATING EMPLOYEES WITH DISABILITIES IN THE WORKPLACE Presented to Mr. Christopher Lee Chief Executive Officer Fanshawe College Prepared by Dana Alhassan Human Resources Manager MEMORANDUM TO: Dr. Christopher Lee, Chief Executive Officer FROM: Dana Alhassan, Diversity Management Manager SUBJECT: Accommodating Employees with Disabilities in the Workplace DATE: December 1, 2014 As part of Fanshawe College’s diversity initiative, here is the report that I conducted to assess the accommodation of employees with disabilities (EWDs) in our workplace. This study includes a review of employers’ misconceptions about hiring persons with disabilities (PWDs), the benefits of including EWDs in the workplace, and recommended workplace accommodations for EWDs. The results of this study show that although employers often claim to have a diverse workplace, most employers are not very proactive in hiring PWDs and hold beliefs about EWDs that research has proven to be incorrect. Based on the competitive advantage EWDs can offer our institution, we should make a considerable effort to accommodate them. The action plan outlined in this report reflects these advantages and suggests accommodations for EWDs that will benefit our institution and all of its employees and students. It is my hope that this report will provide you with the information required to demonstrate the importance of accommodating EWDs in our institution.Show MoreRelatedCanadian Human Rights Case Study735 Words   |  3 PagesRegulations and Resources Available - Ankeen Relevant Legislation It is crucial for employers and Deaf and Hard-of-Hearing employees to understand and comply with the relevant government legislation in terms of Employment. 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HR departments deal directly with everyone employed by a company. HR can include many ethical pitfalls that can lead to a bad reputation or damage a companies financial sustainability if the situation is notRead MoreEmployee Is Injured On The Job1353 Words   |  6 Pagesregular duties. EKU attempts to return employees to work as quickly as medically possible after an occupational injury. In order to reasonably accommodate restrictions, it is sometimes necessary to assign an employee to a position outside of their regular department. This document describes policies and procedures to be used when an employee is subject to modified or restricted duty by a workplace injury or occupational illness. (All references to â€Å"workplace injury† hereafter also apply to work-related

Expression and Purification of rGFP from E. coli free essay sample

The Expression and Purification of Recombinant Green Fluorescent Protein (rGFP) From E. coli strain, BL21(DE3), Using Ni2+-Agarose Affinity Chromatography Abstract: The purpose of these series of experiments was to express and purify recombinant Green Fluorescent Protein (rGFP) from the E. coli strain, BL21(DE3) by beginning with its purification via a Ni2+-agarose affinity chromatography column. The His6 tag of the rGFP bound to the Ni2+-agarose column and washes and elutions were obtained, with elution 3 containing the most amount of fluorescence at approximately 12,000 RFUs. From here, a Bradford assay was performed in order to determine how much protein was in each sample and an SDS-PAGE/Coomassie Blue analysis was done to determine the size and purity of rGFP in the elution 3 sample. The sample came out to be about 34 kDa and was about 75% pure. Lastly, a Western Blot was performed with binding of primary and secondary antibodies to prove that the protein of interest, rGFP, had indeed been expressed and purified. Introduction: Osama Shimomura first discovered Green Fluorescent Protein (GFP) in 1962. The discovery was made inside the bioluminescent jellyfish A. equorea victoria. GFP was found to be a protein of 238 amino acids and had a molecular weight of 26,888 kDa. He was later able to characterize the structure of one of GFP’s unique features- its chromophore, which causes the protein to fluoresce. Later in 1985, Douglas Prasher became the first person to clone GFP (Prasher, 230). For these experiments, a recombinant form of GFP that contained a Histidine6 tag (His6 tag) on the N-terminus of the protein, as well as an X-press epitope tag that begins at the 24th amino acid residue was taken. The His6 tag’s metal ion binding feature was critical during the Ni2+-agarose affinity chromatography because it was able to bind to the nickel inside the column and thus allowed rGFP to be purified since other proteins would be washed out. The purpose of these series of experiments was to allow the expression and purification of rGFP so that further analysis could be done, such as how to determine the purity, size, and total yield of a protein. Finding out this information would give a better understanding of the characteristics of GFP and its significant properties that could lead to further scientific and medical discoveries. Materials and Methods: (Referenced from the â€Å"Biochemistry Lab Manual†) Expression of rGFP in E. coli- Using a single bacterial colony of strain G (BL21pLysS, pRSETA-GFPuv strain from E. coli), 10 ml of LB media [100ug/ml Amp; 25ug/ml Cam] was inoculated and was grown overnight at 37 °C with vigorous shaking until the culture was saturated. Then 500 ml of liquid LB growth media [100ug/ml Amp; 25ug/ml Cam in 1 liter baffled flask, pre-warmed-30 °C] was inoculated with ~4ml of the saturated overnight culture so the culture had an OD600 of 0.1. This culture was grown overnight at 37 °C with vigorous shaking until OD600 of 0.5 was reached. A bacterial pellet designated â€Å"G0† was obtained at time equals zero by pelleting 1ml of culture and discarding the supernatant. The culture was then induced with IPTG (1mM final concentration) and allowed to grow. After 3 hours, a â€Å"G3† bacterial pellet was obtained and both G0 and G3 were stored at -20 °C. Also, 15 ml of the culture was pelleted and the supernatant was discarded, leaving a â€Å"G3-15ml† pellet and was stored at -20 °C to be used later as the starting material to isolate rGFP. Purification of rGFP using Ni2+-Agarose Affinity Chromatography- The rGFP crude extract was prepared before performing the Ni2+-agarose affinity chromatography. The freeze-thaw method was used on the G3-15ml bacterial pellet. The slow freezing at -20 °C causes small ice crystals to form within the bacteria, damaging the bacterial membrane. Addition of 500ul of breaking buffer [10mM Tris, pH 8; 150mM NaCl] twice to the G3-15ml pellet and immediately pipetting the breaking buffer up and down causes the pellet to thaw and the cytoplasm of the bacteria to be released, creating a homogeneous solution. After vortex for 5 minutes, the solution was placed in a 37 °C water bath for 10 minutes. It was then transferred to a rotating platform shaker in a dry air 37 °C incubator for 20 minutes. Once the bacteria had lysed, it was centrifuged at 14,000xg, 4 °C, for 10 minutes. The supernatant was decanted into a new tube and labeled, GCE (rGFP crude extract). The Ni2+-Agarose column was made bypacking in a small amount of glass wool inside a 3 ml syringe. Once secured onto a ring stand with a clamp, a luer-lock was fastened onto the end of the syringe. 2ml of breaking buffer was added to the column and a few drops were excreted before closing the luer-lock to prevent air bubbles. With at ~500ul of breaking buffer in the column, 1ml of 50% Ni2+-agarose slurry was added to the column and the luer-lock was opened to pack the agarose matrix, creating a bed volume of 500ul. The column was then washed with breaking buffer in order to take out any ethanol that the agarose had been stored in. Next, 1ml of GCE was applied and given 5-10 minutes for the his6 tag on the rGFP to bind with the Ni2+-agarose matrix. The luer-lock was then opened to allow ~0.5ml of the effluent to flow into a tube and labeled, W1. The next 0.5ml of the effluent that came out was labeled, W2. Then, breaking buffer was pipetted into the column in 0.5ml increments and each was collected in separate tubes and labeled W3-W10. After the column was rinsed with 5ml of breaking buffer, an elution buffer [10mM Tris, pH 8.0; 150mM NaCl, 300mM imidazole] was added in 0.5ml increments and each of these were collected and labeled E1-310. Determining Protein Concentration of rGFP Fractions- A Bradford assay was used to determine the total protein amount in washes 1-6 and elutions 1-6. First a Bradford standard curve was created using 6 different amounts of Bovine Serum Albumin (BSA)- 0ug, 2.5ug, 5ug, 10ug, 15ug, and 20ug. Each sample was made from a 1mg/ml BSA stock solution and mixed with enough water to bring the volume to a total of 50ul in each tube. 1ml of Bradford reagent was added to each and mixed by vortexing and then incubated at room temperature for 10 minutes. Using a microplate reader, the absorbance at 595nm of each sample could be found. By plotting the data [Absorbance (595nm) vs BSA (ug)], the standard curve was created. The same procedure was done for the W1-6 and E1-6 samples by taking 50ul of each sample and adding 1 ml of the Bradford reagent. Once absorbance was measured, extrapolation of these absorbance values on the standard curve could be done in order to determine the amount of total protein that was present in each sample. SDS-PAGE/Coomassie Blue Analysis- The SDS-PAGE gel was created with 12% resolving gel (used 4X stacking buffer) and 5% stacking gel (used 4X resolving buffer). Both gels contained water, 30% Acrylamide, 10% APS, and TEMED. Using a device made of 2 pieces of glass, the resolving gel was poured in and was solidified before adding the  stacking gel on top. Once it polymerized, a comb was inserted to create the wells in which the samples would be placed. This device was then put inside an electrophoresis tank, which was then filled with electrophoresis buffer. Once the samples (GO, G3, GCE, W3, W4, E3, E4) were prepared with 4X sample loading buffer, they were placed inside the wells and electrophored for 45 minutes at 200 volts. The gel was then removed, stained with Coomassie Blue, and de-stained so that the size and purity of each sample containing the rGFP protein could be determined. SDS-PAGE/Western Blot Development- The SDS-PAGE gel was created, the same prepared samples were loaded, and the electrophoresis was run the same way. This time, the gel was placed inside a cassette (transfer apparatus) with filter paper being placed at the base, then the nitrocellulose (NC) membrane, then the gel, and more filter paper before placing the lid on top. Once the proteins migrated onto the NC membrane, it was stained with 20ml of Ponceau S and de-stained in order to determine the efficiency of the transfer, the orientation of the blot, and to mark the ladder. The blocking step was performed by adding 30ml of 5% non-fat dry milk/TBS solution and incubated for 30 minutes. The membrane was then washed by adding of 30ml of 0.05% Tween 20/TBS and incubated (3X). It was then probed with 7ml of mouse IgG anti-Xpress epitope MAb solution and incubated. The same washing step was repeated 3 times before probing the membrane with 7ml of Sheep IgG anti-mouse IgG conjugated horseradish peroxidase polyclonal anti-serum solution and incubated. After a final wash, 7ml of TMB substrate solution was added, and the membrane was incubated until the desired color intensity was achieved. Results: The expression of rGFP undergoes a process that begins with the induction of IPTG. If no IPTG is induced, the lac repressor will inhibit expression of T7 RNA Polymerase. IPTG inhibits this lac repressor so that a large enough amount of T7 RNA Polymerase is made to overwhelm the inhibition by lysozymes. In figure 1 it is seen that T7 RNA Polymerase allows the T7 promoter in the plasmid to transcribe and translate the rGFP gene. Ampicillin is also to the plasmid to maintain selectivity and protect it from contamination so that only rGFP is being expressed. The T7 promoter will allow the T7 RNA Polymerase to bind to the His6-Xpress-GFPuv tag that’s next to the T7 Promoter and then expression of rGFP will begin. At time equals zero, our G0 sample was collected and then 3 hours after induction with IPTG, our G3 was collected. Both samples were measured qualitatively (handheld UV lamp) and quantitatively (spectrofluorometer). Looking at figure 2, a closer look at the rGFP gene was analyzed. The results showed that the entire gene was 279 amino acids long and contained the His6 tag near the N-terminus, the Xpress epitope tag between the His6 tage and the rGFPuv, and finally the rGFPuv near the C-terminal. Inside the rGFPuv, is the chromophore structure that causes the fluorescence. Since the molecular weight of a single amino acid is 120 Daltons, the MW of this protein can be calculated by multiplying its number of amino acids (279) by the MW of each amino acid (120) giving 33,480 Daltons. A combined activity/elution profile for rGFP was made in order to compare the amount of fluorescence with the total amount of protein in each sample (W1-6, and E1-6) (figure 3). The graph showed the washes to have larger amounts of protein, as was expected since all the undesired proteins were being rinsed out. And although the elutions fewer amounts of protein, they had a greater amount of RFU’s, which indicated being on the right track to knowing whether rGFP had been purified. In the next experiment, an SDS-PAGE gel was created and stained with Coomassie Blue (figure 4). Between elutions 2 and 3, the third one looked the most prominent and therefore was assumed to be the rGFP band. It was located around the 34 kDa marker on the ladder, which is fairly close to the calculated MW of the rGFP protein. Other bands smaller than this were also visible, but at this time could not be identified as a contaminant or rGFP with C-terminus degradation. The band looked to be about 75% pure. In the final experiment, a Western Blot was developed in order to determine whether the proteins had transferred onto the NC membrane and if it was indeed the rGFP protein (figure 5). Undesired proteins were blocked with 5% non-fat dry milk/TBS solution, and then the proteins left were probed with the primary antibody. This primary antibody should have bound to the Xpress-epitope tag in rGFP and when probed with the secondary antibody, it  should have bound on several spots of the primary in order to amplify the signal. After washed with TBS substrate solution, the Western Blot proved that rGFP had been successfully purified and that it was the band at 34 kDa. The Western Blot also showed that the smaller bands were merely contaminants since they did not show up in the final product. Conclusion/Discussion: The attempt to express and purify rGFP from these series of experiments was successful. The Ni2+-Agarose affinity chromatography purified the protein by allowing other proteins to be washed out and rGFP to be collected in the elution samples. The qualitative (handheld UV lamp) and quantitative (spectrofluorometer) measurements taken showed some fluorescence in the washes and this was probably due to the N-terminus (containing the His6 tag) being degraded and therefore not being able to bind to the column. The third elution (E3) ended up having the most amount of rGFP and this was proven with the Bradford assay. The SDS-PAGE/Coomassie Blue Analysis experiment then helped to identify the purity and size of the rGFP in this third elution. With the purity being approximately 75% and the band showing up around the 34 kDa marker, the conclusion was made that rGFP was indeed purified by the Ni2+-Agarose column and that the calculated molecular weight was similar to what was shown on the gel. However, there were some bands at a lower molecular weight that did show up on the gel, but at this time it could not be determined whether they were contaminants or rGFP that had had their C-terminus degraded. In order to determine that the band found in E3 was in fact rGFP, the SDS-PAGE process was done again and this time stained with Ponceau S and a Western Blot was performed. The primary antibody was able to bind to rGFP’s unique feature, the Xpress-epitope tag, and then the secondary antibody amplified and conjugated it. Because the antibodies were able to bind and the visible markings showed up in the Western Blot, this was able to prove that the bands on the gel were indeed rGFP. The fact that they didn’t bind to the lower bands with the smaller molecular weight brought on the conclusion that there were simply contaminants that did not contain the Xpress-epitope tag. After these conclusions were made, follow-up experiments could be performed to see how useful GFP could be. One experiment that could be done is taking the gene that encodes this green fluorescent protein and transfecting the cells of another organism to see if it could produce fluorescence as well. Another experiment could be taking GFP and using it as a marker to tag a protein of interest to see this protein’s activity and its’ localization in the cell (Chalfie, et. al.). Experiments with GFP could prove to be very helpful in the world of science by discovering the expression and activity of proteins and how they can affect living organisms. References: Rippel, Scott. Biochemistry Lab Manual. Print. Prasher, Douglas C., Virginia K. Echenrode, William W. Ward, Frank G. Prendergast, and Milton J. Cormier. â€Å"Primary Structure of the Aequorea Victoria Green-Fluorescent Protein.† Gene 111 (1992): 229-233 Chalfie, Martin. â€Å"Green Fluorescent Protein as a Marker for Gene Expression.† National Center for Biotechnology Information. U.S. National Library of Medicine. Web. 04 Aug. 2013. http://www.ncbi.nlm.nih.gov/pubmed/8303295. Figure 5. Western Blot of rGFP Fractions. Figure Captions Figure 1. Plasmid Map of rGFP from E. coli strain, BL21(DE3). IPTG inhibits the lac repressor protein allowing T7 RNA polymerase to be produced. The PT7 promotes expression on His6-Xpress-GFPuv and placed in a media that contain ampicillin to maintain selectivity so that only rGFP is produced Figure 2. The schematic diagram of rGFP from the E. coli strain, BL21(DE3). The numbers indicate where each amino acid sequence begins. The Histidine-6 tag begins at the 5th amino acid residue. The Xpress Epitope tag begins at the 24th amino acid residue. Finally, the rGFPuv sequence, being 238 residues long, begins at the 39th amino acid residue. It also contains the chromophore (65-67 residues of rGFPuv, Ser-Gly-Tyr) which is located from the 103rd-105th residue. This diagram displays the entire sequebce of the rGFP protein, coming out to be 279 amino acids long. Figure 3. Combined activity/elution Profile of rGFP during washes 1-6 and elutions 1-6 in a Ni2+-agarose column. The graph shows RFU’s (left) and total protein in ug (right) for each wash and elution. Once the rGFP had been purified in the Ni2+-agarose column, a breaking buffer (10mM Tris, pH 8.0; 150mM NaCl) was used to create the washes by pipetting the buffer in 0.5ml increments and collecting each in separate tubes labeling them W1-W6. Then, an elution buffer (10mM Tris, pH 8.0; 150mM NaCl; 300mM imidazole) was collected in increments of 0.5ml and labeled E1-E6. We could measure the amount of RFU’s each sample had using 200ul of each fraction and placing them in a spectrofluorometer. E3 showed to have the greatest amount of fluorescence. Using the Bradford assay, the total protein of each sample was determined by extrapolating points from the standard BSA curve. Both values were scaled up. Figure 4. SDS-PAGE gel/Coomassie Blue of rGFP fractions from E. coli strain, BL21(DE3). The gel was created by using 12% resolving gel which was composed of water, 4X resolving buffer (0.75M Tris, pH 8.8, 0.4% SDS), 30% Acrylamide (29.2% w/v acrylamide, 0.8% w/v bis acrylamide), 10% APS (ammonium persulfate), and TEMED (tetramethylenediamineAsds). Once solidified, a 5% stacking gel composed of water, 4X stacking buffer (0.25M Tris, pH 6.8, 0.4% SDS), the same 30% Acrylamide, 10% APS, and TEMED was added. Once the stacking gel was polymerized, the gel was ready to be loaded with fractions of the E. coli strain, BL21(DE3), that we collected from previous lab. A molecular weight marker (ladder) was also loaded onto the gel. Going left to right, lanes 1and 2 are disregarded, the G3, G0, GCE, W1, W2, E2, E3, ladder. The gel was placed in a gel electrophoresis tank and filled with electrophoresis buffer (450ml H2O, 50ml 10X stock solution (30g Tris, 144g glycine, 10g SDSD per liter). Figure 5. Western blot of the rGFP samples. An SDS-PAGE gel was created with 12% resolving gel and 5% stacking gel and ran through the electrophoresis procedure. The gel was then placed in a cassette and on top of a nitrocellulose membrane so that the protein could transfer from the gel to the membrane. The Ponceau S staining identified the presence of rGFP. It was then blocked with 5% non-fat dry milk/TBS solution and probed with a primary antibody, mouse IgG anti-Xpress epitope MAb solution. It binds uniquely to the Xpress-epitope tag in rGFP so it can be isolate. It was then probed with the second antibody, Sheep IgG anti-mouse IgG conjugated horse radish peroxidase polyclonal anti-serum solution. This would amplify and conjugate the rGFP and finally, it was washed with TMB substrate solution to create the color intensity desired.