Reality and Illusion in Shakespeares Hamlet - Appearance versus Realit

Appearance versus Reality in Hamlet   â Shakespeare's play, Hamlet, is a story of a youthful sovereign who must discover reality with respect to his dad's passing. All through the play, the major topic of appearance versus the truth is consistent. Most of the principle characters take cover behind shroud of falsehoods and duplicities, darkening reality to the point that almost nothing of their genuine selves are noticeable. The maze of misleading is bent to such an extent that lone Hamlet knows about reality, and simply because the phantom of his dad uncovered it to him. Hamlet, Polonius, Rosencrantz, Guildenstern, and the King Claudius are all piece of this hover of trickery.   â â â Hamlet, while more authentic than the rest, carries himself into the misleadings with his faked craziness. In any event for this situation there is an advantageous avocation; his every activity and word is accounted for legitimately to Claudius by Rosencrantz, Guildenstern, Polonius or any number of others faithful to Claudius. His madness is a cunning strategy for security; he will be disregarded and free as long as he isn't viewed as a danger. In spite of the fact that utilizing a considerable amount of double dealing, Hamlet's wrongness is little in contrast with that of Polonius, the illustrious associate.   â â â Polonius is fixated on anticipating the pictures of a trusting and liberal dad and an insightful man by and large, controlling or misleading everybody conceivable to serve his own plan. One way he endeavors to improve his picture is the point at which he over and again waxes lovely and conveys extensive talks with respect to life to his youngsters. A case of this is the point at which he is giving Laertes his approval to leave for France and gets done with this hopeful guidance, This most importantly: to thine own self be valid./And it must follow... ... A.C. Statement. Abstract Companion to British Authors: William Shakespeare. San Diego: Greenhaven, 1996. Danson, Lawrence. Disastrous Alphabet. Modern Critical Interpretations: Hamlet. Ed. Harold Bloom. New York City: Chelsea House Publishers, 1986. 65-86 Findlay, Alison. Hamlet: A Document in Madness. New Essays on Hamlet. Ed. Imprint Thornton Burnett and John Manning. New York: AMS Press, 1994. 189-205. Hopkins, Lisa. Parison and the Impossible Comparison. New Essays on Hamlet. Ed. Imprint Thornton Burnett and John Manning. New York: AMS Press, 1994. 153-164. Rose, Mark. Improving the Role. Modern Critical Interpretations: Hamlet. Ed. Harold Bloom. New York City: Chelsea House Publishers, 1986. 117-128 Wiggins, Martin. Hamlet Within the Prince. New Essays on Hamlet. Ed. Imprint Thornton Burnett and John Manning. New York: AMS Press, 1994. 209-226.

Men and Women Essay Example | Topics and Well Written Essays - 500 words

People - Essay Example Additionally, they likewise have similitudes and contrasts regarding passionate and mental requirements which will be managed in detail. Right off the bat, people have comparable physical needs, for example, food, attire and sanctuary. The two sexual orientations need food and water so as to live. They likewise need garments not exclusively to decorate themselves yet particularly to get them far from the perils of nature, for example, the risky impacts of the warmth of the sun or from the cold during winter. Asylum is likewise important for the two people to keep them secure. Besides, they have comparable enthusiastic needs, for example, the should be adored. Since affection is supposed to be the general language, it is obvious that individuals around the globe paying little mind to race, age and sexual orientation need this passionate worth that is by all accounts normally a piece of each person. What's more, people experience torment, demoralizations and other antagonistic passionate stressors that influence the manner in which they live. In this way, they additionally need feelings that calm the agonies, for exampl e, bliss, affirmation and supportive gestures to lift their spirits and empower them to defeat the troubles life brings. Thirdly, people additionally have comparable mental needs. Since they have comparative feelings of trepidation and concerns, people need comparable mental impedances. For example the two people stress over what they look like so the two of them should be guaranteed that they look great. At the point when they have accomplished something, the two people should be applauded for their prosperity or they will feel like they are failures. On the other hand, there are additionally contrasts among people that make them unmistakably unique in relation to one another. First among these distinctions would be their physical needs. For example, ladies are known to be increasingly worried about what they look like thusly, they invest more energy and cash on their attire, extras and make up. They invest a great deal of energy contemplating how they would show up with the goal t hat ladies frequently wind up spending

What are the Pros and Cons of Cigarette Smoking

What are the Pros and Cons of Cigarette Smoking Addiction Coping and Recovery Personal Stories Print The Pros and Cons of Smoking A List Compiled by an Ex-Smoker By Terry Martin facebook twitter Terry Martin quit smoking after 26 years and is now an advocate for those seeking freedom from nicotine addiction. Learn about our editorial policy Terry Martin Updated on January 07, 2020 Mixmike / Getty Images More in Addiction Coping and Recovery Personal Stories Methods and Support Overcoming Addiction Alcohol Use Addictive Behaviors Drug Use Nicotine Use As of Dec. 20, 2019, the new legal age limit is 21 years old for purchasing cigarettes, cigars, or any other tobacco products in the U.S. Most  long-term smokers have a love/hate relationship with cigarettes. From the moment we awake in the morning until we lay our heads down on the pillow at night, cigarettes punctuate each and every activity of our daily lives. It becomes a very heavy load to carry over time. When we decide to quit, untangling the associations weve built up over a lifetime of smoking takes conscious effort;?? something that smoking cessation forum member Zoe illustrates beautifully below. In her list of pros and cons, Zoe takes a critical look at how smoking made her feel. A powerful exercise in stepping out from behind the smoke screen that nicotine addiction forces us to live behind, a pros and cons list allows us to uncover the truth about our relationship with smoking. From there, the work of healing can begin ... as it did for Zoe. From Zoe I made a list of what I liked about smoking vs. what I hated about smoking ... and though I really missed it at first, looking at this list made me see that I didnt like smoking as much as I thought I did. What I Liked About Smoking The bonding I experienced with other smokers.The feeling of creating a ritual.Watching the cigarette burn and watching the smoke swirl.Momentary gratification. What I Hated About Smoking The after-smell on my clothes, furniture, car, house, everything. Yuck.Not being able to breathe properly.The constant nagging cough. All day, all night.Lots of phlegm, lots of throat-clearing and losing my voice mid-sentence.Painful heartburn every night and every time I drank coffee.Feeling winded after extremely mild activity.Severe throbbing headaches, occasional migraines.Lingering colds and bronchitis.Racing heartbeat, more sweating.Increased rate of hypertension.Dizziness after smoking too fast or [having] too many cigarettes.Nausea from smoking too much.The constant coppery, ashy taste in my mouth.Yellow skin, teeth, and fingernails.Scaly, unhealthy-feeling skin.Anxiety from the fear about what I was doing to myself and the consequences.No relaxation, always feeling in need of something. A constant feeling of not being satisfied.Mini-withdrawals throughout the day.Feelings of shame while spending time with nonsmokers.Not accomplishing tasks because of wasted time smoking.The late-evening/middle-of-the-night trip to the gas station.Going out in bad weather to smoke alone.Feelings of inadequacy and substance dependence.Driving my cat out of the room every time I lit up.Dry mouth and constant feelings of thirst.Coughing so hard that I made myself sick.Trembling hands and fingertips.Fear. Of being unable to quit, of dying an untimely, painful death.The stinging feeling in my lungs when I tried to take a deeper or slower breath.Getting smoke in my eyes.Burning my lips on the filter.Trying to light short butts and feeling my eyebrows singe. Ouch!Re-lighting a previously torched cigarette, so I dont waste any tobacco.Overflowing ashtrays, ashes, and dust everywhere.Burn holes in my car upholstery and on my clothes.Will I fall asleep smoking?Will I catch something on fire?Dry, chapped lips.The cost. All that money wasted on ruining my health and well-being.My nails and hair grew very slowly.Smoking fueled my compulsiveness relating to other bad habits, such as nail-biting and binge-eating.Having to reapply my lipstick after smoking.The filthy taste of cheap tobacco.Having to crack the car window in the pouring rain. Wet leg, wet arm, water in my eyes.Tar build-up on windows and furniture.The way my hair and skin smelled.Limited motivation and energy.Spilled tobacco in my purse, on my dresser, on my computer desk.Lighting the filter end by mistake...Dropping a cigarette while driving.Trying to tap my ashes out the car window ... while the window is rolled up.Dropping hot ashes or losing the tip of a cigarette.Oops! Tapped ashes in my drink.Feeling exiled in the smoking section/smoking room.Dulled sense of taste and smell. Maybe you should sit down and make a list like this for yourself. It might give you the nudge towards where you know you want to be.~Zoe A Word From Verywell Zoe is right. Crafting your own list of pros and cons is an eye-opening way to see just what smoking means to us, good, bad and ugly. A list will help you build motivation to quit smoking once and for all. Think about how smoking makes you feel, both physically and emotionally.  Try to honestly list out all of the positives and negatives and it will reinforce your desire to stop the madness that smoking is.?? The resources below will help you understand what to expect as you move through the process of recovery from nicotine addiction. Why You Should Consider Quitting SmokingWhat to Expect from Nicotine WithdrawalPersonal Quit Stories Dont fear smoking cessation.  Dig your heels in and go.  The discomforts are all temporary and give way to outstanding benefits that will continue to grow with time.

Practical Strategies To End Procrastination - Free Essay Example

Sample details Pages: 3 Words: 986 Downloads: 9 Date added: 2019/06/14 Category Psychology Essay Level High school Topics: Procrastination Essay Did you like this example? In Part 1 of this series, we shared insight into the causes of procrastination. We discovered how procrastination often disguises itself in an alternative activity. We observed how habitual procrastination could result in chronic stress. Lastly, we focused on Three Essential Tips to program your mind to override the tendency to procrastinate. If you missed this valuable part of the series, you can check it out here. In todayrs post, we are going to share 6 Practical Strategies that boost your motivation . . . To start your tasks as soon as possible, To accomplish your tasks on time or even earlier, To tackle your tasks with maximum effort and energy! Don’t waste time! Our writers will create an original "Practical Strategies To End Procrastination" essay for you Create order Practical Strategies to Permanently End Procrastination Practical Strategy #1: Time Yourself Timing your tasks has become an extremely popular strategy for overriding procrastination. Itrs easy to apply and it really gets you pumped to accomplish your task. Herers how it works: You need a timer. The timer is your new boss. The timer rules no exceptions. First, you set the timer for one task, ideally 20-30 minutes .You work continuously on the one task without any diversion whatsoever. When the time rings, you re-set it for 5 10 minutes. During this time, you take a break. Step away from your desk. Do something completely different: eat a snack, play music, look out the window, exercise, watch TV. Just make sure whatever you do is not work-related. When the timer rings again, re-set it for another 25 minutes. Immerse yourself in the single-task focus for the set time. Then re-set the timer for another 5 10 minute non-work related break activity. This is known as the Pomodoro technique, named after the originator. Each set of work and break times is known as one Pomodoro. Aim for four Pomodoros and you will see your tasks completed. If you finish the task before the set time, then begin the next one until the timer rings. On the other hand, if 30 minutes seems too short, then stretch it out. The magic is the timer. Its outside influence is very strong and energizing much more so than just thinking about the time in your mind. The single-minded focus on the task relieved by short breaks forces you to get the job done while the breaks prevent fatigue. Have fun with this one! Practical Strategy #2: Reward Yourself Nothing motivates people more than a reward at the end of a long and tedious task. You dont have to indulge in expensive gifts. Treat yourself to a movie, a walk in fresh air, or a special snack. Stick to simple, pleasurable activities your due compensation for not procrastinating. Soon youll discover your type of rewards that motivate you on even the most challenging tasks. Practical Strategy #3: Set Punishments Now for the flipside, perhaps a little draconian . . . If you reward yourself when you do not procrastinate, then should you punish yourself when you do procrastinate? Self-imposed discipline will keep you on track with good work habits and lower stress levels. Here are some types of self-imposed discipline: Avoid watching TV the whole day Avoid Facebook for 24 hours Anonymously send a lot of money to someone whom you dislike with all your might Obviously, these punishments are meant to keep you on track so you take your work seriously. Focus on what needs to be done at all times and I guarantee you will never have to face your own punishments. Practical Strategy #4: Involve Other People Dont try to accomplish this all by yourself. Countering the habit of procrastination is a battle with the mind. Invite someone to monitor your actions a close friend, co-worker, or relative, as suits the situation. You are accountable to that person. If you dont meet your deadline, they should admonish you! IMAGE Let that happen and be sure to take it seriously. they are your support network. Beating procrastination can be difficult without the help of family and close friends. So, dont take your support network for granted! Maybe you can do the same for them at some time. Practical Strategy #5: Change Your Beliefs and Values About Time Time is like cash; when you spend it frivolously or haphazardly, itrs gone forever. And it always disappears too soon! You can never recover those hours you wasted on unimportant activities. What you can do: Start changing your beliefs and values about time. Your time is a scarce and essential resource. View it that way. Respect and honor it. Spend it wisely. Are you spending a lot of time on unimportant, time-consuming activities? If so, you must make the conscious choice either to limit or even avoid those activities altogether. Get in the habit of asking yourself, Is this the best use of my time at this moment? Do it now so you dont commit the same mistakes in the future. We already know what happens when a person runs out of time to complete all the things that he needs to do anger, frustration, regret. So, exercise the foresight to avoid this unnecessary stress. Practical Strategy #6: Assert Your Right To Use Time Wisely Some people feel pressured or obligated to acquiesce to any and all invitations from friends, family and coworkers. Sure, itrs healthy to socialize. But not when it impinges on your schedule. You make this determination. Remember: Time rules. And it doesnt wait. You have to learn how to say no to unimportant invitations. You know best when you need your time for more important tasks and obligations. If your associations are accustomed to passive compliance with visiting, you can expect them to react with questions and even negative attitude. So, just be sure to explain the reason youre declining some activities that you used to attend regularly. People who truly know and care about you will immediately understand what youre trying to do. As for the rest If they fail to appreciate your self-help efforts, do they really deserve any your time?

Intolerance in American Society Essay - 629 Words

Intolerance in American Society Intolerance of other peoples culture, religion, ethnic background and skin colour was a major issue in the 1920s America. This was, of course the era of the economic boom that helped to make America a rich and prosperous country in which to live. This economic boom made America a very attractive place to immigrate to, especially to those in countries that were not so well off. This meant immigration on a massive scale. People from all over the world, particularly Eastern Europe came together in one city and work for a living. The amount of people from different countries and social backgrounds all in one small area i.e. a city, America is huge, is bound to†¦show more content†¦These areas where a place that was reserved for your kind of person and if overstepped the line and moved in to a street where you were not of the ethnic majority then you would expect a cold, even fierce reception. These ghettos were in virtually every northern city with blacks being mainly in Chicago and New York. Racial tensions were not only present in the northern cities; they were all over the southern states as well as black people sometimes outnumbered the whites. The first black people came to America as slaves by the white settlers in the 17th century. By the abolishment of slavery in the 1800s, blacks did indeed outnumber white people in southern states at least. The white government, scared of their potential power introduced freedom-binding laws primarily aimed at black people. They were aloud to vote by national law, but local authorities prevented this in most cases, they had no access to a decent education, jobs and lived in poverty well in to the modern age. As well as harsh laws against blacks, white supremacists set up an organisation called the Ku Klux Klan. This Klan discriminated and used violence against black, sometimes lynching them etc. The KKK was a powerful political force throughout the 1920s. Examples of KKK hatred are being beaten to a pulp for being a foreigner and marrying an American girl and a black man being lashedShow MoreRelatedTelevision Interview : The Crucible 1118 Words   |  5 Pagesgreat emphasis on his reputation in society. ABC: As in most plays and movies, there are important themes represented. What are some of these in the movie? D: Well, there are several themes that we can relate to even in our modern times, such as themes of intolerance, fear, hysteria, reputation, integrity, greed and jealousy, revenge, pride and authority. ABC: Can you tell us more on some of the themes as it relates to today’s societies? D: ‘The Crucible’, written by ArthurRead MoreVincent Chin and Post-9/11 Victims: Travesty of Justice1514 Words   |  6 Pagesis a country of immigrants, as a popular saying goes. 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Dissertation on Histone Protein Segregation Free Essays

Introduction Histone Proteins are favourably the alkaline proteins which are present in the nucleus of the eukaryotic cell. They are the basic parts responsible for wrapping and organizing DNA into chromosomes in the nucleus. They are the principal protein elements of the chromatin and acts as a spool around which the DNA winds. We will write a custom essay sample on Dissertation on Histone Protein Segregation or any similar topic only for you Order Now In the nucleus of the cell the DNA is arranged in a very dense and super coiled style to form chromosomes. This is referred to as DNA packaging. (Isenberg, 1979) The unwound DNA in the chromosomes is very long and cannot be fit in the nucleus of the cell, hence these histone proteins brings about the DNA wrapping thereby reducing the dimensions of DNA in the cell. (Alberts B et al., 2001) Histones are primarily categorized into five major types. They are H1/H5, H2A, H2B, H3 and H4. They are categorized into two classes. They include core histones with H2A, H2B and H3 and linker histones with H1 and H5. Two of each of core histones unites to form one octameric nucleosome which is the fundamental sub unit of chromatin. (Bartova et al. 2008; Bonisch et al. 2008). Hence histones proteins form the nucleosomes directly. After extraction of chromatin from the cells it is like beads on string. The string is the DNA and the beads are the nucleosomes which are roughly disc shaped. The linker histone H1 separates the nucleosome with the other nucleosomes and also helps in bringing the adjoining nucleosomes for further super coiling. The octameric nucleosome is formed by two H2A, H2B dimers and a tetramer of H3, H4 histones. These core histones are comparatively analogous in structure. However their 3D structures are relatively different. The core histones have different protein sequence. The sequence for H2A Histone is SGRGK QGGKA RAKAK SRSSR AGLQF PVGRV HRLLR KGNYS ERVGA GAPVY LAAVL EYLTA EILEL AGNAA RDNKK TRIIP RHLQL AIRND EELNK LLGRV TIAQG GVLPN IQAVL LPKKT ESHHK AKGK. (Andreas and David, 1998) The sequence for H2B Histone is PEPAK SAPAP KKGSK KAVTK AQKKD GKKRK RSRKE SYSIY VYKVL KQVHP DTGIS SKAMG IMNSF VNDIF ERIAG EASRL AHYNK RSTIT SREIQ TAVRL LLPGE LAKHA VSEGT KAVTK YTSSK. (Andreas and David, 1998) The sequence for H3 Histone is ARTKQ TARKS TGGKA PRKQL ATKAA RKSAP ATGGV KKPHR YRPGT VALRE IRRYQ KSTEL LIRKL PFQRL VREIA QDFKT DLRFQ SSAVM ALQEA CEAYL VGLFE DTNLC AIHAK RVTIM PKDIQ LARRI RGERA. (Andreas and David, 1998) The sequence for H4 Histone is SGRGK GGKGL GKGGA KRHRK VLRDN IQGIT KPAIR RLARR GGVKR ISGLI YEETR GVLKV FLENV IRDAV TYTEH AKRKT VTAMD VVYAL KRQGR TLYGF GG. (Andreas and David, 1998) The molecular weight of the core histone proteins are measured in the Daltons. The theoretical molecular weight for H2A histone protein is 13,990.28 Daltons, for H2B histone protein is 13,788.97 Daltons, for H3 histone protein is 15,272.89 Daltons and for H4 histone protein is 11,236.15 Daltons. (Kornberg, 1977;McGhee and Felsenfeld, 1980) The histone proteins play a significant role in chromosome stabilisation, gene regulation and expression. The main functions of histone proteins are condensing the DNA strands and chromatin regulation. These histone proteins form the nucleosome and they impart a structure to which DNA is twisted and causes to fit the bulky genomes of eukaryotes inside the nucleus of the cell. (Bartova et al. 2008; Bonisch et al. 2008). The histones also undergo post translational modifications which alter their interaction with DNA and nuclear proteins. The H3 and H4 histones have very long tails projecting from the nucleosome which can be covalently altered at numerous places and the alteration includes the acetylation, ubiquitination, methylation, phosphorylation, ADP-ribosylation and citrullination.(Bartova et al. 2008; Bonisch et al. 2008). The modification of histone includes the gene regulation, DNA repair and chromosome condensation. In this manner, histones can control gene expression, cell g rowth and proliferation. (Jenuwein and Allis 2001). The separation of the histone proteins is carried out by using the 1D SDS PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis) method. In the gel electrophoresis method the charged molecules are segregated based on their physical properties like charge, mass, etc. by allowing them to pass forcedly through a gel matrix by an electric current. (Coligan et al., 2002).The proteins from the various complex protein mixtures are normally separated by this method by using the polyacrylamide gel. It is known as Polyacrylamide Gel Electrophoresis (PAGE method). In this method the main constituent is the acrylamide which is used in the preparation of the electrophoresis gels for the isolation of proteins. In order to form the cross linked polymer network, the acrylamide is combined with bisacrylamide with the polymerising agent like Ammonium Per Sulphate (APS). The polymerisation reaction is catalysed by TEMED (N,N,N,N’-tetramethylenediamine) by the producing the free radicals by APS. The size of the pores and firmness of the gel matrix mainly depends on the ratio of bisacrylamide and acrylamide used and their total con centration. Sequentially these in turn depends up on the range of molecular weight of the proteins which has to be determined. The pore size of the gel matrix is contrary to the quantity of acrylamide used. Generally 12% of polyacrylamide gel has lesser pore size than 7% of polyacrylamide gel. Larger proteins are resolved with the gels with less amount of acrylamide and smaller proteins with more amount of acrylamide. And for the broad range of protein sizes, the special gels like Gradient gels are prepared by having less percent of acrylamide at the top/start and more percent of acrylamide at the bottom/end. The electrophoresis gels are mixed with the buffers which impart the conduction of electric current though the matrix. The solution is transferred to the gel cassette which is a thin space formed by placing two glass or plastic plates facing each other. After the gel is polymerised, the cassette is placed vertically into the electrophoresis tank containing the electrodes. The p roteins are added in the wells from the top and electrophoresis is carried out during which the proteins are separated by the gel due to its sieving properties. In order to obtain the best possible resolution of the proteins a gradient gels are used. (Hames et al., 1990) There are different forms of PAGE available which are used for separation of different proteins based on different principles. They commonly include the native PAGE, SDS PAGE, 2D PAGE, etc. (Hames et al., 1990; Coligan et al., 2002). In case of the 2D PAGE (Two Dimensional Polyacrylamide Gel Electrophoresis) the proteins are isolated by isoelectric point in the first dimension and later by mass in the second dimension. This method offers the maximum resolving power for the protein analysis and it is a very significant method in proteomic research. (Hames et al., 1990) In the native PAGE, the proteins are isolated based on their size, net charge and shape. The electrophoretic migration is due to the fact that the most of the proteins possess a net negative charge in alkaline running buffers. As the negative charge density increases, the migration speeds of the protein increase. Simultaneously the frictional force of the gel matrix produces a sieving effect thereby reducing the protein movement based on their size and shape. Hence the smaller proteins face less frictional force and larger proteins face more frictional force. In this manner the various proteins are separated from a mixture. (Hames et al., 1990) In case of the SDS PAGE the gel is mixed with the buffer having the SDS (Sodium Dodecyl Sulphate). The SDS is heated with the proteins samples before electrophoresis so that the charge density of all proteins are almost equal. The SDS is an anionic detergent which denatures the proteins present in the sample and attaches strongly to the uncoiled molecule. In order to make sure that no quaternary or tertiary proteins structure remains generally a reducing agent like dithiothreitol (DTT) is also added as it ruptures the protein disulphide bonds. Hence when these samples are used in the electrophoresis, the proteins get separated based on their mass alone. In this method a set of proteins of known molecular weight is run aside the sample in the same gel cassette. They act as a reference from which the mass of the sample proteins is determined. They are called as Molecular Weight Markers. The electrophoresis is carried out by using the two gels for favourable results. They are the stacki ng gel and the resolving gel. The stacking gel is added over the top of the resolving gel. Apart from the low concentration of acrylamide in stacking gel, it has low pH and different ionic content than the resolving gel. This causes the proteins to get concentrated into a tight band during the early electrophoresis period before entering into the resolving gel. (Hames et al., 1990) Breast cancer refers to the hysterical growth of breast cells. The breast carcinoma is a malignant tumour. It occurs due to any abnormal genetic changes in the breast cell. Presently there is an increasing alarm regarding the high risk posed by various compounds with oestrogen like activity present in the environment. These various compounds includes the Phytoestrogens (e.g. genistein), food products like legumes, lentils, chickpeas, soybean, cereals, fruits, and vegetables, industrial contaminants, (e.g. bisphenol A) and polychlorinated biphenyls, organochlorine pesticides (e.g. endosulfan), etc. These substances have the ability to produce cancer mediated through estrogenic receptors. Some of these substances when treated with the MCF-7 cell line, MCF-10F (ER/ER?+), MDA-MB-231 (ER/ER?+) or MCF-10A (ER/ER) cells, increased the growth rate of these cell lines. This 3-4 folds proliferation was due to the up regulation of all the core histone proteins which represents the increase in t he chromatin content of the cells. The degree of proliferation of the cells indicates the level of the core histone proteins and was concentration dependent. Hence this indicates that histone proteins are used as indirect markers of breast cell proliferation. Therefore in the human breast cancer the histones are up regulated and cause cell proliferation mediated by the oestrogen receptors agents. Based on this it is possible to state that the core histone proteins serve as bio markers of (ER+) human breast cancer.(Zhu et al., 2009) Aim The main aim of this experiment is to resolve the given four human recombinant protein samples (H2A, H2B, H3 and H4) by using the one dimensional SDS Poly Acrylamide Gel Electrophoresis (1D SDS – PAGE). Material and Methods Equipments used The experiment uses the Bio Rad Mini PROTEAN 3 Cell gel electrophoresis unit. It consists of the electrophoresis tank, electrode chamber with a cathode and anode, two glass plates, gel cassette assembly, comb and the connecting cables. Power source, heater, micro centrifuge, pH meter, and the other general equipments like test tubes, conical flasks, etc were used. Reagents used The various reagents used in this experiment includes the Acrylamide/bisacrylamide (30%w/v/0.8%w/v), 3.0M Tris/HCl (pH – 8.8 for resolving gel stock), 0.5M Tris/HCl (pH – 6.8 for stacking gel stock), TEMED, SDS (10%w/v), Ammonium Per Sulphate (APS – 25%w/v). The running buffer is also used as 0.025M Tris/0.192M glycine/0.1% (w/v) SDS, pH 8.3. The other reagents used are sample buffer, Pre stained molecular markers, colloidal Coomassie Blue Stain Solution, Bromophenol Blue (0.5%w/v) and the given four human recombinant proteins (1Â µg/Â µl) [H2A, H2B, H3.3 and H4]. Experimental Procedure The SDS PAGE method is commonly used for the examination of the proteins because of its simplicity, speed and resolving capacity. The SDS PAGE is the Sodium Dodecyl Sulphate Poly Acrylamide Gel Electrophoresis method. The various steps performed are as follows: Preparationof Gel Cassette Sandwich Firstly select a clean Spacer Plate and Short Plate and place them over one another and set them in the casting frame and lock the pressure cams from both the sides. It should be kept in such a manner that the short plate should face the front of the frame. After locking the gel cassette assembly check that both the plates are flush at the bottom. Then place the rubber gasket at the bottom of the casting stand and place the gel cassette sandwich assembly in the casting stand in such a manner that it is perpendicular to the level surface. After complete formation of the gel cassette assembly, add water into it to make sure that no leaking takes place. Then remove the water and wipe it with the filter paper. Set the comb into the assembled gel cassette and mark the level at 1 cm below the comb teeth and remove it, the resolving gel is poured up to this level. After preparation of the resolving gel pour it up to the mark into the assembled gel cassette in a very smooth way to prevent an y air bubble formation. Allow the gel to set for 45 minutes. After the gel has set wash its surface with distilled water and wipe with the filer paper and allow drying the area in between the glass plates for a minute. Then after this prepare the stacking gel solution and pour it above the resolving gel till the top of the plates. Then immediately place the comb in the stacking gel in gel cassette to form the wells. Allow the stacking gel to set for overnight. Preparation of Gels The gels used in SDS PAGE method are the resolving gel and the stacking gel. Both the gels consists of the acrylamide/bisacrylamide, distilled water, 10% SDS, TEMED and ammonium per sulphate (APS). However the resolving gel uses 3.0M Tris/HCl (pH 8.8) and the stacking gel uses the 0.5M Tris/HCl (pH 6.8). These gels are prepared by directly mixing all the reagents. However the TEMED and APS are added only when the gel is ready to be polymerised. In this manner the gels are prepared. Electrophoresis Module Assembly After the stacking gel has set, smoothly remove the comb. Then open the cams and remove the sandwich gel cassette and place it in the electrode assembly in such a manner that the short plate faces inward of the electrode assembly. Secure the electrode assembly by closing the two levers of the frame to form the inner chamber. Set this secured inner chamber into the Mini Electrophoresis Tank. Fill the inner chamber with the running buffer, however it is not overfilled. Then add around 200 ml of the running buffer is added into the Mini Tank. Sample Preparation and Loading Four recombinant histone protein samples are used in this experiment. They are H2A, H2B, H3.3 and H4. 1Â µl of each of this protein samples (1Â µg/Â µl) is added to 20Â µl of the Sample Buffer in the Eppendorf tubes and labelled properly. They are heated in a heater set at 100? C for two minutes and then allowed to cool to room temperature. After the sample preparation, 20Â µl of each of the histone protein is loaded into the wells carefully by using the gel loading tips. The sample loading should be very smooth and the tip should not puncture the well. Load the 2Â µl of the pre stained molecular marker (Page Ruler) into the other well simultaneously to ensure the movement of proteins. Gel Electrophoresis After loading the histone proteins, mini tank is covered with the lid and the electric supply is given by the cable wires. Turn on the power supply and the start the gel electrophoresis at 200 Volts for about 35 minutes until the bromophenol blue dye has drifted to 1 cm from the bottom of the gel. During this time each histone protein based on their molecular weight is moved to their ends. Staining the Gels Once the electrophoresis is completed, switch off the power supply and take out the lid. Cautiously lift the inner chamber assembly and dispense the running buffer to avoid the falling of buffer before opening the cams. Then open the cams and take out the glass cassette sandwich. Open the sandwich with the help of gel releaser and take out the gel slowly. Immerse the gel in 30 ml of Colloidal Coomassie blue stain solution for half an hour with moderate shaking. During this time the staining occurs and once it is completed the gel is de stained with water and then incubated with water at room temperature for overnight for complete de staining. After this the gel is rinsed with water for the proteins bands to be seen clearly. Results Figure 1: SDS Poly Acrylamide Gel Electrophoresis of Histone Proteins. Bands show the migration of individual histone proteins. A pre stained molecular marker is included at the side for comparison. In the present experiment four human recombinant histone proteins were given for isolation and determination. From the standard pre stained molecular marker, each of the histone protein has to be determined by correlating with its band. The Figure 1 illustrates the SDS Poly Acrylamide Gel Electrophoresis of given four recombinant histone proteins (H2A, H2B, H3.3 and H4). In the figure the last column of bands refers to the standard pre stained molecular marker (Page Ruler) and the other four bands represents the respective histone proteins. The values beside the molecular marker indicate the molecular weight in kDa. Hence with reference to this standard values, the four given histone protein samples has to be determined. In case of the band of H2A histone protein, it is nearly 13,990 Da as it is below 15 kDa. Similarly in case of the band of H2B histone protein, it is nearly 13,789 Da. And in case of H3.3 histone protein, the band is clearly at the top of all the bands indicating it has the highest value and it is slightly above 15, so it may be around 15,273 Da. In the fourth histone protein H4, the band is at the far end when compared to the other three bands. However it is slightly above 10. Hence it may be around 11,236 Da. Therefore the molecular weights of the histone proteins were identified. However the standard molecular weight for the H2A is 13,990.28 Da, for H2B is 13,788.97 Da, for H3.3 is 15,272.89 Da and for H4 is 11,236.15 Da. (Kornberg, 1977;McGhee and Felsenfeld, 1980) Discussion The separation of proteins is mainly performed by the gel electrophoresis method. There are different types of gel electrophoresis methods used to separate different types of proteins. However in the present experiment the isolation of histone proteins is mainly carried out by 1D SDS PAGE method. This method is a one dimensional method and it separates the proteins principally by their molecular weight. It makes use of the ionic detergent Sodium Dodecyl Sulphate (SDS). The poly acrylamide gel is mixed with SDS because it denatures the proteins and binds to them to make them approximately evenly negatively charged. This is done with the aid of heating and generally a reducing agent like dithiotheritol (DTT) in order to break the protein disulphide bonds. This removes the risks of presence of any tertiary or quaternary proteins. As a result when electric current is passed through a power supply, all the SDS bound proteins of the sample will drift in the gel matrix to the anode, thereby separating the proteins based on their molecular weight alone. The proteins with low molecular weight moves fast through the gel compared to the proteins with high molecular weight due to the sieving effect of the gel matrix. Therefore in this experiment a set of four human recombinant histone proteins (H2A, H2B H3.3 and H4) were given to separate them based on their molecular weight. Hence these protein samples were taken and were subjected to the SDS PAGE method. In this method firstly the gel cassette assembly was formed by using the two glass plates (Spacer Plate and Short Plate) in a proper manner such that the short plate is facing front of the frame. After assembling the gel cassette it was filled by the gels which were prepared just before use. Firstly the resolving gel was poured and after it got set for about 45 minutes, the stacking gel was poured and comb was placed in it and it was allowed to set for overnight. The pouring of gels was carried out very carefully and slowly in order to prevent any air bubble formation. The appropriately set gel with the well defined wells is selected and gel cassette assembly was set in the electrode unit in the Mini Electrophoresis Tank. The running buffer was added properly and the sample histone proteins were loaded very cautiously. Before loading the protein samples, they were heated for about two minutes. Along with the samples, the pre stained molecular marker was also loaded. And the power supply was given and the electrophoresis was carried out for 35 minutes at 200V. During this time the bromophenol blue dye moves to 1cm from the bottom of the gel. After this the power supply was switch off and very carefully the gel was removed from the gel cassette sandwich assembly and was dipped in the staining solution like Colloidal Coomassie blue stain solution for about half an hour with occasional shaking. After staining, the gel is removed and de stained with distilled water by incubating it for overnight. Then it was rinsed properly to visualise the bands for each of the histone proteins. The pre stained molecular marker of known molecular weight was also run beside the given histone samples in the same gel. It acts as a standard or reference. For all the given four human recombinant histone proteins the bands lie in between the 10 and 15 kDa as per the marker. In situation of H2A histone protein, the band obtained is just below the 15 kDa band in the standard. Hence its molecular weight is around 14 kDa. In case of the H2B histone protein, the band is obtained also below 15 kDa but a little lower than H2A, hence it should be less than 14 and should be around 13.7 kDa. In case of H3.3 histone protein, the band is just above the 15 kDa and it should be around 15.3 kDa and for the H4 histone protein the band is quite low and just above the 10 kDa band; hence it should be around 11.3 kDa. The data obtained in this experiment is fairly correct as compared to the standard theoretical data. The theoretical molecular weight for H2A histone protein is 13,990.28 Daltons, for H 2B histone protein is 13,788.97 Daltons, for H3 histone protein is 15,272.89 Daltons and for H4 histone protein is 11,236.15 Daltons. (Kornberg, 1977;McGhee and Felsenfeld, 1980). Hence the results obtained in this experiment are reasonably acceptable and can be used for further studies. If the mixture of these histone proteins were need to be separated by the same method (SDS PAGE), then the expected result would not be accurate and proper. And the band for the individual histone protein may not be clear. It is because all the given four human recombinant histone proteins have approximately equal and very close molecular weights. And the values for all the four histone proteins lie between 10 to 16 kDa. Hence the bands would be very close to each other. If any flaws or any possible inaccuracy occurs during the experiment, then there is a possibility that the bands may be wrong. Hence the results would be wrong. Therefore there is very less possibility that the results would be accurate and also for the correct identification of the bands. However, if the experiment is carried out perfectly, then in such case the order of bands would be H4, H2B, H2A and H3.3 in the increasing order of their molecular weights. It is resolved with the help of the standard molecular mar ker which acts as a reference. Therefore, if mixture of these four histone proteins were to be separated by this method very accurately, then the results may be correct. However, for the separation of this mixture of four histone proteins another laboratory method is available. In this method, the groups (H2A, H2B) and (H3, H4) are separated first by using NaCl and hydroxylapatite chromatography. (Richard and Gary, 1979) The H2A and H2B group are the slight lysine rich group and H3 and H4 is the arginine rich group. This method was introduced by Van der Westhuyzen and Von Holt. In this method, firstly the individual groups were separated. However, the given mixture was treated with 0.63 M NaCl and 0.1 M potassium phosphate before the mixture sample is loaded on to the hydroxylapatite. This is to remove any contamination with the other linker histone proteins like H1 and H5. Then it was subjected to chromatography to obtain the core histone groups (H2A, H2B); (H3, H4). After this the samples obtained are subjected to SDS PAGE method for further separation. Finally the core histones were separated. (Richard and Gary, 1979) References Ahmad, K. and Henikoff, S. (2002). Histone H3 variants specify modes of chromatin assembly Proc. Natl. Acad. Sci. U. S. A. 99 Suppl 4, 16477-16484. Andreas, D.B. and David, L. (1998 Oxford University Press). Nucleic acids research. Histone Sequence Database: new histone fold family Vol. 26, No. 1, 372-375. Bartova, E. Krejci, J. Harnicarova, A. Galiova, G. and Kozubek, S. (2008). Histone modifications and nuclear architecture: A review J. Histochem. Cytochem. 56, 711-721. Bonisch, C. Nieratschker, S.M. Orfanos, N.K. and Hake, S.B. (2008). Chromatin proteomics and epigenetic regulatory circuits. Expert Rev Proteomics.5, 105–119. Coligan, J.E., et al. , Eds. (2002). Electrophoresis, In Current Protocols in Protein Science, John Wiley and Sons, Inc. New York. 10.0.1-10.4.36. Hames, B.D. and Rickwood, D. Eds. (1990) Gel Electrophoresis of Proteins: a Practical Approach, 2nd ed. Oxford University Press, New York. Isenberg, I. (1979). Histones. Annu. Rev. Biochem. 48, 159-191. Jenuwein, T, and Allis, C.D. (2001). Translating the histone code. 293. 1074–1080. Kerenyi, L. and Gallyas, F. (1973). Errors in quantitative estimations on agar electrophoresis using silver stain Clin. Chim. Acta 47, 425-436. Kornberg, R. D. (1977). Structure of chromatin Annu. Rev. Biochem. 46, 931-954. Marino-Ramirez, L. Jordan, I. K. and Landsman, D. (2006). Multiple independent evolutionary solutions to core histone gene regulation Genome Biol. 7, R122. McGhee, J. D. and Felsenfeld, G. (1980). Nucleosome structure Annu. Rev. Biochem. 49, 1115-1156. Richard, H.S. and Gary, F. (February 1979). Nucleic acids research, oxford journals. A new procedure for purifying histone pairs H2A + H2B and H3 + H4 from chromatin using hydroxylapatite Volume 6 Number 2, 689-696. Ruchel, R. Steere, R. L. and Erbe, E. F. (1978). Transmission-electron microscopic observations of freeze-etched polyacrylamide gelsJournal of Chromatography A 166, 563-575. Shapiro, A. L. Vinuela, E. and Maizel, J. V.,Jr. (1967). Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels Biochem. Biophys. Res. Commun. 28, 815-820. vanWert, J. M. Wolfe, S. A. and Grimes, S. R. (2008; 2008). Binding of RFX2 and NF-Y to the testis-specific histone H1t promoter may be required for transcriptional activation in primary spermatocytes J. Cell. Biochem. 104, 1087-1101. Weber, K. and Osborn, M. (1969). The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis J. Biol. Chem. 244, 4406-4412. Zhu, Z. Edwards, R. J. and Boobis, A. R. (2009). Increased expression of histone proteins during estrogen-mediated cell proliferation Environ. Health Perspect. 117, 928-934. Version 112,113, 114,115 and 116. UniProtKB/Swiss-prot release 2010_04 P16104 How to cite Dissertation on Histone Protein Segregation, Essays

The Trojan Women A monologue from the play by Euripides Essay Example For Students

The Trojan Women A monologue from the play by Euripides Essay A monologue from the play by Euripides NOTE: This monologue is reprinted from The Plays of Euripides in English, vol. i. Trans. Shelley Dean Milman. London: J.M. Dent Sons, 1920. ANDROMACHE: Hear, that with pleasure I may touch thy soul Not to be born, I argue, and to die, Are equal: but to die is better far Than to live wretched; for he knows not grief Who hath no sense of misery: but to fall From fortunes blessed height, to the low state Of abject wretchedness, distracts the soul With the keen sense of former happiness. Like as the light of life she neer had seen, Polyxena is dead, and of her ills Knows nothing: I, who aimed at glorious rank, And reached my aim, from fortune widely erred: All that to prudent matrons gives a grace, In Hectors house was ever my employ. First, for in this to women blame is due, Charged or not charged, to such as rove abroad, I checked this wandring humour, and remained At home, within my house; nor gay discourse Of females there admitted, but intent On ordering what was useful, deemed myself Well occupied. With silence of the tongue And cheerfulness of look I entertained My husband: where my province to command I knew, and where to yield obedience to him. The fame of this was bruited through the host Of Greece, and wrought my ruin; for the son Of fierce Achilles, soon as I was made A captive, wished to take me as his wife, Doomed in the house of those, whose slaughtring hands I rue, to be a slave. From my fond heart Could I rend Hector, and expand my breast To this new husband, faithless to the dead Should I appear: if I disdain his love, I shall excite the malice of my lords. Short time, they say, to a new lord disarms A womans hate: but her my soul abhors, Who for new nuptials slights her former husband, And loves another: een the social steed, Divided from its fellow, draws the yoke Reluctant; yet the beast, by nature formed Less excellent, nor speech nor reason knows. O my loved Hector, I was blest in thee, Thou was the lord of all my wishes, great In understanding, noble birth, and wealth, And valour: from my fathers house thou first Leddst me a virgin to the bridal bed: Now thou are perished, and I mount the bark For Greece, a captive to the servile yoke. Hath not the death then of Polyxena, Whom thou bewailest, lighter ills than mine! For not to me een Hope, which still is left To all of mortal race, remains; no thought That better fortune eer will visit me With pleasing expectation cheats my mind. We will write a custom essay on The Trojan Women A monologue from the play by Euripides specifically for you for only $16.38 $13.9/page Order now