Analysis Of Purified R Gfp Using Sds Page Gel/Coomassie Blue And Western Blot

2803 words - 11 pages

ABSTRACTGreen fluorescent protein (GFP) has great potential for biological studies due to its stability, flexibility, and unique characteristics. The GFP gene can be cloned and reconfigured to be expressed in numerous organisms such as E. coli to induce its expression for purification and analysis. The rGFP produced can then be purified using Ni+²-agarose column and quantified using Bradford assay. Purity analysis of the rGFP obtained was done using SDS-PAGE gel/Coomassie Blue. Finally a Western Blot was performed to confirm the protein present is actually the protein of interest, rGFP. According to these different methods, yield, purity and rGFP activity were found and they were 4.20 ug, 15% and 10200 RFUs, repetitively.INTRODUCTIONGreen fluorescent protein, also known as GFP, is a protein produced by a jellyfish Aequorea Victoria which fluoresces in the green portion of the visible spectrum. GFP is an extremely stable 27kDa protein composed of 238 amino acids, which form a ß-barrel wrapped around a central core. The intrinsic fluorescence of the protein is due to a covalently attached chromophore which is formed by posttranslational intramolecular reactions involving cyclization and oxidation of amino acids Ser – Tyr – Gly [1]. By combining and incorporating the multiple mutations (such as one class of GFP fold and cyclize properly and another class do not )will lead to an increased in fluorescence.Poly histidinetag (6xHis tag) is an amino acid motif in proteins that consists of at least six Histidine (His) residues, often at the N- or C-terminus of the protein [3]. Histidine tagging allows affinity isolation of the protein with attendant proteins without affecting the function of the protein. In order to purify the rGFP protein, the gene fusion technique was used in which the GFP protein was fused with another protein (poly histidinetag) that is easily purified by Ni+2 –agarose affinity chromatography. The tagged protein (rGFP) has affinity for the metal ion nickel, hence it binds to the Ni+²-agarose matrix in the column, and the other impurities that are not bound to the column are washed away. When rGFP is passed through a column containing immobilized nickel, it binds to nickel through His residues. Then the tagged protein is eluted by imidazole which competes and displaces the His-tag from nickel co-ordination, freeing the His-tagged proteins.Through experiments, the expression and purification of rGFP was done successfully. The ease in detection is shown in its ability to fluoresce up to 509nm when excited with ultraviolet light of 395nm [1]. No cofactor is required for fluorescence, which makes it inexpensive for experiments. Real time detection is made possible by shining UV light on the fractions, which will then fluoresce green color to indicate the presence of rGFP. Since GFP can be cloned and configured (recombinant GFP) to be expressed in numerous organisms such as E. coli, only small amounts of...

Find Another Essay On Analysis of Purified rGFP Using SDS-PAGE gel/Coomassie - Blue and Western Blot

Why Spaghetti Westerns Should Simply Be Westerns: An analysis of the Western Genre using examples from "The Searchers" and "For a Few Dollars More"

1930 words - 8 pages Why Spaghetti Westerns Should Be Simply WesternsIt is argued that true Westerns all conform to the same mold of the prewar Westerns: A lonely hero who wanders the West looking to settle down, but is never able to give up the violent life he has led. John Cawelti says the Western takes place on the border between two lands, between two eras, and with a hero who remains divided between two value systems (Altman, 220). Schatz describes the Western

Analysis Of Miles Davis' "Kind Of Blue"

623 words - 2 pages In early 1959, jazz trumpeter Miles Davis laid down the foundation for a whole new style of jazz music. Through his "Kind of Blue" modal jazz was born. This record became a classic, at times showing its complexity through the soloing, but also allowing the educated listener to revel in the simplicity of the modes. Davis planted the seeds for this new style in his album "Milestones" but "Kind of Blue" showed that the style had matured and was

Benefits and Services of Blue Cross Blue Shield of Massachusetts

2167 words - 9 pages Blue Cross Blue Shield of Massachusetts (Blue Cross), employees are offered a broad range of benefits and services, which are nonfinancial payments for continuing their employment with the company. At Blue Cross, there are a majority of benefits and services that are exceptional as well as those that a few that need improvement.Blue Cross provides health care coverage to 2.9 million members and it takes more than 3,800 associates to work around

Holden, Charlie, and Devon: An Analysis of the Psychological Disorders in Three Characters INCLUDES OUTLINE AND WORKS CITED PAGE!

2154 words - 9 pages OUTLINEHolden, Charlie, and Devon:An Analysis of the Psychological Disorders in Three CharactersTo truly understand the characters (as well as their thoughts and actions) in The Catcher in the Rye, The Perks of Being a Wallflower, and Not as Crazy as I Seem, it is necessary to understand the psychological disorders that they deal with, such as manic-depressive disorder, obsessive--compulsive disorder, Post-traumatic Stress disorder, and others.A

Essay of Short Fiction story by Native American author Tom Whitecloud. This essay describes the setting for "Blue Winds Dancing" using MLA format and quotes from the story

1005 words - 4 pages , mountains, snow, the "blue winds" and his people mean to him. When he reaches his homeland he is very happy to be home.This is the opening of the story. Whitecloud is tired. He wishes he has spent more time with his people:There is a moon out tonight. Moon and stars and clouds tipped with moonlight. And there is a fall wind blowing in my heart. Ever since this evening, when against a fading sky I saw geese wedge southward. They were going home

An Appeal to Action: Rhetorical Analysis of Change.org’s Launch Page

1405 words - 6 pages people’s support. For a simple payment, Change.org offers companies a faster method to petition to a wider audience than what the company could achieve unaided. At the bottom of the page, Change.org further assures users and companies that it deserves their trust by quoting several relevant and popular news sources. These sources include TIME Magazine, US News and World Report and Venture Beat. Quotes include: “Change.org has emerged as one of

The Western Genre: An Analysis of its History and Rise and Fall as a Genre

935 words - 4 pages WESTERN GENRE 10 (2007) both portray darker and twisted forms of the conventions from the Classic Western. Loyalty, trust, and moral rights are hard to distinguish in films like these. These Revisionist Westerns played an important role in keeping the Western genre alive by bringing any Western convention from the past and using it to create an interesting narrative. References Campbell, N. (2013). Post-Westerns. University of Nebraska Press

The Life of Alan Shapiro and Analysis of “Country Western Singer”

1051 words - 5 pages , 2009; for his book which is entitled Old War. Shapiro’s poetry is unlike many others to say the least if Shapiro’s poetry is read assumptions can be made that Shapiro is depressed or that he endured a very difficult life while he was young. His poem Country Western Singer happens to impose this sense of tragedies and doubtfulness and just simply not caring as the poem explains the stages of a man becoming an alcoholic until his last drop of

Strategic Analysis of Jet Blue Airlines: Executive Summary, Financial, Internal and External Industries, Competitors, Substitutes, and Stategies

7722 words - 31 pages Strategic Analysis of Jet Blue AirlinesExecutive SummaryJetBlue is a company built on a focus strategy of low-priced, no-hassle ticketing and refreshingly efficient customer service. The company began with the goal to eliminate many of the complexities and asininities of commercial air travel and set a new standard for customer service. Thus far the company has flown beyond these goals and everyone's expectations while returning a handsome

Isolation, hydrolysis and characterization of casein from non-fat milk and protein analysis using Bradford method

3769 words - 15 pages test, and the Hopkins-Cole test. In the protein analysis, Bradford method is the method used to determine the concentration of protein in the sample. The experiment aims to isolate and purify casein from non-fat milk by isoelectric precipitation. The purified protein samples are subjected to alkaline hydrolysis. The functional groups and specific amino acids present in the protein isolates and hydrolyzates are characterized and determined using

Analysis of The Blue Hotel by Stephen Crane

578 words - 2 pages Analysis of The Blue Hotel by Stephen Crane "The Blue Hotel" by Stephen Crane is a story about three travelers passing through Fort Romper, Nebraska. Pat Scully, the owner of the Palace Hotel, draws the men to his hotel that is near the train station. In the hotel the three men meet Johnnie, son of Scully, and agree to play a game of cards with him. During the game, the Swede declares Johnnie as a cheater; this gives rise to a fistfight

Similar Essays

Sds Page Electrophoresis Of Proteins Essay

1650 words - 7 pages AimsTo analyze four protein samples (Crude extract, AS1, AS2, AS3) using native (non-denaturing) polyacrylamide gel electrophoresis (PAGE).To identify a number of proteins and isoenzyme bands by staining with Coomassie Blue and isoenzyme staining and to determine their relative mobilities.IntroductionThe resolution of separate components in a protein mixture is obtained most clearly using an electrophoretic method. It is used to characterize a

Beta Galactosidase And Western Blot Essay

4002 words - 16 pages acquired from the extraction process performed on both cultures of E.coli strains, EMG26 and EMG 9, which were later subjected to sodium docecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Moreover, inspection of the topmost band on the eighth and the ninth lane revealed the presence of beta-galactosidase, the only protein that can be detected in the gel stained by coomassie blue.2. Inspection of the nitrocellulose paper revealed the

The Use Of The Western Blot Analysis To Identify Four Unknown Species Of Fish

1373 words - 6 pages Protein extractions from unidentified fish samples were separated according to the molecular weights by SDS polyacrylamide gel electrophoresis. Since some of these proteins are shared between fishes, phylogenetic evaluation was reached. Western blot analysis was used to identify four unknown species of aquatic animals via comparison of actin/myosin bands. According to the results of this assay, the best estimate is that the unidentified aquatic

Using Pcr And Gel Electrophoresis To Determine Genotype

772 words - 3 pages Using PCR and Gel Electrophoresis to Determine Genotype In certain situations, it is necessary to identify DNA retreived from a sample. When there is a small sample in need of identification, Polymerase Chain Reactions are used to multiply the DNA in the sample in to many identical samples. The DNA retrieved from the reaction can then be imported into an aparatus using gel electrophoresis to compare the sample of DNA to other samples