LDH Purification lab Report Essay Example

LDH Purification lab Report Paper OLD was purified from the ammonium sulfate precipitated protein mixture by affinity chromatography and its activity was studied by spectrophotometers determination of NADIA at 340 NM. From Pierce BCC assay of crude homogenate, initial protein concentration was shown to be 100 MGM/ml. The final protein concentration of the pooled affinity sample was shown to be 0. 2 MGM/ml. It was found that the total specific activity of OLD was 58. 5 mol/min/MGM, and yield of 0. 6%. Even though we were successful in purifying OLD enzyme, further steps can be taken to increase the yield. Materials and Methods Cell Lysine and Extraction of OLD: Approximately 40 g of minced chicken breast eat (40. 327 g) is blended with ml cold extraction buffer in four 30-seconds bursts for homogenate of the muscle tissue. The extraction buffer contained mm Tries-HCI (pH-7. 4), mm 2-Merchantable, mm Phenylmethylsulfonylflouride (AMPS), 1 mm Ethylene Dianne attracted acid (EDIT). The homogeneities procedure was carried out in the cold room to prevent the denomination of proteins. The homogenate was centrifuged at 15,000 RPM for 20 minutes at 40 C. The supernatant was filtered through two layers of cheesecloth to remove lipids from the supernatant. The total volume was noted and three 0. Ml aliquots (crude extract) were stored at -200 C. Ammonium sulfate precipitation: 60% ammonium sulfate concentration was used to precipitate proteins. 0. 39 g of ammonium sulfate per ml of the supernatant was added gradually to the supernatant for 15-20 min with continuous gentle stirring at 40 C. The mixture was centrifuged for 20 minutes at 1 5,000 RPM at 40 C. The supernatant was discarded and the pellet was stored at -200 c. We will write a custom essay sample on LDH Purification lab Report specifically for you for only $16.38 $13.9/page Order now We will write a custom essay sample on LDH Purification lab Report specifically for you FOR ONLY $16.38 $13.9/page Hire Writer We will write a custom essay sample on LDH Purification lab Report specifically for you FOR ONLY $16.38 $13.9/page Hire Writer Dialysis: Ammonium precipitation leads to high concentration of salts in protein mixture that can interfere with further purification steps. In order to remove excess salts, dialysis was performed. The pellet was suspended in Tries-AMPS buffer (10 rim Tries-HCI, pH 8. 6, 0. 5 mm 2-Merchantable, and mm ratio of EDIT) and mixed very gently until it dissolved at 40 C. Volume of ml protein mixture was added in the dialysis tubing and incubated twice overnight with two IL buffer changes (Same buffer as extraction buffer that was used for cell lysine). After two incubation, protein mixture was responded gently and centrifuged for 10 minutes at 15,RPM at ICC. Pellet was discarded, total volume of supernatant was noted and three 0. 1 ml aliquots were collected. Affinity Chromatography: Isobaric Blue column was used to separate OLD from the other proteins. Ml fractions were collected in thirteen test tubes. Column was first rinsed with Tries-AMPS buffer followed by addition of protein mixture. Then, ml AND Buffer (mm Tries-HCI pH-8. 6, 0. Mm 2-Merchantable, mm Lithium acetate and 1 mm AND+) was added followed by NADIA (mm Its- HCI PH 8. 6, mm NADIA and 0. Mm 2-Merchantable). Between each steps, column was washed with ml Tries-AMPS Buffer. Each fraction was subjected to absorbency reading of Mann. For absorbency above 1. NM, 1:10 dilutions were carried out. Activity Assay: We used OLD Enzyme assay to measure the amount of OLD activity in our protein mixture. OLD catalysts the conversion of lactate to private and AND+ to NADIA. The NADI A can be determined spectrophotometers at 340 NM. The OLD assay was performed in the crude homogenate, desalted fraction and six peak fractions from the Isobaric blue column. A cocktail solution was prepared by mixing lactate stock solution (120 rim lithium lactate, 10 mm Tries-HCI; pH 8. 6), AND+ stock solution (12 mm AND+, 10 mm Tries HCI; pH 8. 6) and bicarbonate stock solution (18 mm Enhance, 0. 5 M Nasal) in the ratio of in cavetti. 0 micrometers of the sample is then added and the assay absorption is measured at Mann. If absorbency was above 1. 5, samples were diluted. Protein Assay: The Pierce BCC Protein Assay (Thermo Scientific) is a detergent- compatible formulation based on bioscience acid (BCC) for the colorimetric detection and quantization of total protein concentration. A series of standard solution of Bovine Serum Albumin (BAS) ranging from 0-2000 pig/ml was prepared from a stock solution of 2 MGM/ml BAS. Lull of diluted crude (1:500, 1 :250), desalted (1:100, 1:50), and 6 peak fractions from isobaric blue column (1:10, 1:5) ere loaded in microscope along with lull of BCC working reagent. Microscope was incubated for mini at ICC and then the absorbency was measured at Mann. Results/Discussion The purpose of this experiment was to extract and purify OLD enzyme from chicken muscle tissue using a variety of techniques including homogeneities, ammonium sulfate precipitation, dialysis, and affinity chromatography. Activity and Protein assay were used to track the overall amount of OLD present in the samples. Crude Extraction: Chicken muscle tissue was homogeneity in a blender with cold extraction buffer in order to else cells, releasing OLD into slurry of tissue monuments. Centrifugation separated membranes, nuclei, and other large cellular components to a pellet leaving a supernatant of crude product. Controlling temperature was a major consideration after homogeneities since not only did this step releases proteins like OLD from the cell, but it also releases proteases that can now interact to degrade the OLD. Keeping samples on ice, pre-cooling the buffer, and avoiding excess kinetic energy through conservative blending were methods used to minimize activity of these proteases. After filtration through cheesecloth, our final volume of crude homogenate sample ml, much more volume than expected. Addition of more than ml of buffer volume could have increased the volume. Other possible explanation is that more solid components such as fats were present in the sample and hence, more than 20 minutes of centrifugation was required. Desalted Sample: 60% ammonium sulfate is added to the crude extract that precipitates OLD proteins. The resulting 40% pellet theoretically contains most of the original OLD, which is re-suspended in very less volume (ml) to create a more concentrated sample. This process leads to high concentration of salts in rotten mixture that can interfere with subsequent purification steps. Ml protein mixture underwent dialysis procedure that removes excess salts and our final volume after dialysis was ml. One possible explanation for increase in our volume could be that extraction buffer got mixed with protein mixture either due to tubing leaking or tubing clips not being properly tightened. Affinity Chromatography: Isobaric Blue column is an affinity column, which is specific to dehydrogenate type proteins, due to a compound structurally similar to NADIA being attached covalently attached to the column. 13 fractions were elected and absorbency was measured at Mann to check presence of OLD protein in the fractions. 1:10 dilution was performed if absorbency reading was above 1. NM since it spectrographically indicates saturation and less than 1% light reaching the detector. During the addition of protein mixture (fraction# 4), high absorbency reading of NM was obtained (Fig. 1). This could be due to lot of non-dehydrogenate-type proteins present in our sample that got eluted first during affinity chromatography. Second peak was seen after AND+ was added since AND solution results in the removal of the loosely bound protein. Third peak was seen after NADIA was added since NADIA solution results in release of maximum OLD proteins (Fig l) Enzyme Activity Assay: The OLD activity was measured spectrophotometers by measuring the absorbency of NADIA at 340 NM. Three peak fractions were selected for this assay based on their absorbency values obtained after adding AND+ (fraction# 6, 7, 8) and other three after adding NADIA in the affinity chromatography step (fraction# 10, 1 1 , 12). A huge activity of 141 mol/min/ml was seen at fraction# 7(PUFF ) which indicated that we had lot of proteins present in our sample. Second peak activity was seen t fraction indicating that more OLD proteins is present in this fraction than in fraction# 11 (PUFF) (fig. 1). Based on this information, we selected fraction #10 as for our protein assay. Desalted showed highest activity among all the samples (Tablet ) possible due to errors occurring during dialysis explained previously. Figure 1. Absorbency readings of eluted obtained from affinity chromatography with OLD activity for 6 peak fractions. The desalted fraction was loaded to the Isobaric blue column and proteins were eluted with Tries-AMPS, AND+ and NADIA wash subsequently. The absorbency at 280 NM of eluted were measured after ACH collected fractions. The OLD activity was calculated from the absorbency values obtained at Mann. Protein Assay: We used BCC Pierce Assay to determine protein concentrations in our protein mixture. BAS standard curve was created for series of dilutions ranging from 0-2000 pig/ml and linear graph equation was used to calculate protein concentrations for the samples (Table 1). Based on Table 1, with each subsequent purification step, protein concentration decreases as sample become more concentrated with only OLD protein. Specific activity should increase and total activity should decrease with very purification step as samples get less and less diluted.

The Roman Empire and the Gladiator Games essays

The Roman Empire and the Gladiator Games essays The Roman Empire was the undoubted pinnacle of society prior to the Middle Ages, and nothing came close to expressing its might, culture, and subjects as the Gladiatorial games did. Views on such Roman sports being cruel and barbaric are merely superficial as these events were deeply integrated within this ancient empire, representing its ideals. Often these were spectacular battles in awe-inspiring arenas which were themselves an architectural masterpiece of those times. Above all, the games portrayed the glory of success, the satisfaction of which nothing else could give. To the Romans, from the Emperor to the commoner, their empire was the epitome of power. These games were showpiece events that displayed the might and glory of victory that Romans hold so close to their hearts, especially so, at a time when war and territorial conquests was getting infrequent. As seen in Gladiator, the sight of a victorious competitor in the arena after a bloody battle brought immense cheering and approval from the fans. Only the best gladiators were deemed appropriate to compete in the Colosseum which greatly raised the games' standard. The replication of these games in the provinces served to highlight the influence these games had and indirectly integrated these towns closer to the central axis of power, Rome (referred to as "the light" in the movie). The battle arenas themselves symbolized power in the architectural sense. The Colosseum was the largest and perhaps most elegantly crafted structure in Rome, and it was only fitting that such a magnificent arena sh ould stage important events such as gladiatorial games. With their obsession of power and glory, it is no wonder that the crowds are fickle enough to change "allegiance" to whichever group of gladiators is winning the battles at the moment as seen in the battle between the charioteers and the slave gladiators in the movie. The sponsor of the Games (usually the Emperor) also showed his o...

Aztecs and the Aztec Civilization

Aztecs and the Aztec Civilization Aztecs are the collective name given to seven Chichimec tribes of northern Mexico, who controlled the valley of Mexico and much of central America from its capital during the Late Postclassic period from the 12th century AD until the Spanish invasion of the 15th century. The main political alliance creating the Aztec empire was called the Triple Alliance, including the Mexica of Tenochtitlan, the Acolhua of Texcoco, and the Tepaneca of Tlacopan; together they dominated most of Mexico between 1430 and 1521 AD. For a complete discussion see the Aztec Study Guide. Aztecs and their Capital City The capital city of the Aztecs was at Tenochtitlan-Tlatlelco, what is today Mexico City, and the extent of their empire covered almost all of what is today Mexico. At the time of Spanish conquest, the capital was a cosmopolitan city, with people from all over Mexico. The state language was Nahuatl and written documentation was kept on bark cloth manuscripts (most of which were destroyed by the Spanish). Those that survive, called codexes or codices (singular codex), can be found in some small cities in Mexico but also in museums around the world. A high level of stratification in Tenochtitlan included rulers, and a noble and commoner class. There were frequent ritual human sacrifices (including cannibalism to some degree), part of the military and ritual activities of the Aztec people, although it is possible and perhaps likely that these were exaggerated by the Spanish clergy. Sources An  Aztec Civilization Study Guide has been developed with loads of details on lifestyles of the Aztecs, including an overview and detailed timeline and king list. The photograph used on this page was provided by the Field Museum for a part of their new exhibit Ancient Americas. Also Known As: Mexica, Triple Alliance Examples: Azcapotzalco, Malinalco, Guingola, Yautepec, Cuanahac, Templo Mayor, Tenochtitlan

Coal chemical looping Research Paper Example | Topics and Well Written Essays - 3000 words

Coal chemical looping - Research Paper Example As it has been reported that the global demand for energy is likely to be fulfilled by the exploitation and utilization of fossil fuels in decades to come, the need for developing effective systems for the capture and storage of CO2 has become indispensible (Yue 2010). While, the significance of this observation is undeniable the present systems and frameworks which are utilized extensively in the industry are inadequate and do not support the advancement of certain aims and objectives that are associated with the development of suitable technologies. As noted by Yue (2010), present solutions for solving the issue are marked by the absence of cost-efficiency, capture efficiency and conversion efficiency which is an alarming scenario for both researchers and analysts alike. This observation proposes that the technology of chemical looping combustion (CLC) and its related processes have emerged as an innovation which presents numerous prospects and opportunities for the development of energy and power-related technologies by minimizing the risks, limitations and drawbacks that are associated with the solutions that are currently and presently available within the industry. Numerous studies and researches have established that the CLC technology boosts of impressive efficiency rates for the purpose of producing electricity and hydrogen in scenarios where the system of gasification is associated and integrated within the chemical looping technology (Yue 2010). Since the inception of the innovation, empirical studies have also been able to establish the types of suitable components that should be installed within an effective CLC system to maximize desired outcomes. This aspect involves the selection of solid fuels that can be used in chemical looping in addition with the oxygen carriers which have the potential to enhance the oxidation process. Even though, these observations have revealed the possibility of utilizing an extensive range of carbonaceous fuels and ca rriers, the scope of this paper focuses on the process of coal chemical looping such as the coal-direct looping process and the process of syngas chemical looping. Furthermore, the paper also explores the practical applications of chemical looping combustion in organizations such as General Electric and Alstom. In conclusion the research examines the advantages, challenges and limitations that are associated with the process of coal chemical looping in the light of recent researches and studies. The application of chemical looping combustion (CLC) processes is a revolutionary advancement and innovation in the industry, as stated by Yue (2010): Chemical-looping combustion (CLC) is a new alternative to conventional combustion that prevents the CO2 from being mixed in the combustion gases. This is accomplished by preventing the air-N2 to be present in the part of the reactor system where the oxidation of the fuel takes place. (p. 527) The introduction and subsequent incorporation of ch emical looping techniques and strategies was impelled by the absence of frameworks that could be applied for the separation and conversion of chemicals during the occurrence of various product-related procedures (Fan 2011). While, this observation is reflective of the traditional demands and needs which initially required the launch of chemical looping techniques, contemporary applications of chemical looping essentially aim to fulfill the need of establishing a modern, optimized and effective

Writer's selection Personal Statement Example | Topics and Well Written Essays - 750 words

Writer's selection - Personal Statement Example She reminded me that I would become a resourceful person that everyone in the society would be looking for if I By inculcating good virtues into my lifestyle if I would inculcate good virtues into my lifestyle. As far as picking ‘Communications’ as my major is concerned, I have to admit that I had considered reasons in my mind at the time I was joining Seattle community College. In this respect, I have to mention that my very first reason was to be able to interact with all the diversified persons living in Hong Kong. Fundamentally, Hong Kong is a metropolitan with diverse cultures that are autonomous hence ‘communications’ will play a major role when it comes to meeting new people from various backgrounds. The second reason as to why I chose communications as my major is the fact that I like to get out of the comfort zone. This was particularly evident when visited Los Angeles for 3 weeks in July 2010 as an exchange student. I deemed it essential to learn certain skills of communicating with various personalities. Lastly but more importantly, I picked communications as my major is because I like communicating with people and discovering new things. When it comes to my professional experience, I find it worthy to mention that I have participated in quite a number of initiatives that have added significant value to my personal and professional life. For instance, between January 2013 and March 2013, I volunteered as a teaching assistant at ACRS (Asian Counseling and Referral Services). In that capacity and organization, I acquired excellent interpersonal and interpersonal skills. Moreover, I carried on with the volunteering work from April 2013 to June 2013 Arts Corps. I gained invaluable knowledge during the two-month period that I still hold dear to date. My third place where I gained invaluable experience was at CJR where held the position of an Education Consultant. In this position, I played several roles including

My Dream Job Essay Example for Free

My Dream Job Essay What is a bad boss or manager? We all have had some good managers and some bad managers. In most cases people don’t care too much for their boss. One of the biggest characteristics flaws in a poor manager is poor communications skills. They possess little to no listening skills and don’t seek out advice or suggestions from their employees. When a big decision needs to be made they normally make the decision on their own with little input from their subordinates. Another characteristic of a poor boss is a one who is afraid of confrontation. In management, I believe that any time there is an issue, the boss should not be afraid to address it. Some employees will take advantage and lose respect for their boss if that happens. I call those types of managers â€Å"buddy bosses,† which is not a good thing their needs to be a healthy balance. Bosses should be nice most of the time yet they also needs to be firm when the time calls for it. Good management skills don’t always come naturally. Managers have to continue to have ongoing training on leadership skills just like the workers need to have ongoing training on the jobs. In management; it is hard to please everyone in fact, if everyone is happy with you all the time you are probably a â€Å"buddy boss. † There will always come a time when you are going to have to say no to someone’s request. I believe that sometimes bad employees force a boss to become more firm most of with the employees. There are several types of bosses that I would consider bad. The first one is called an incompetent boss. They really don’t know the job and rely on the employee’s knowledge to get them through. When an issue occurs, they are no help at all. When an incompetent boss tries to help, they often make the situation worse. The second type of poor boss is a unchangeable leader. This is someone who does not like change and is unwilling to do what it takes to make the team more effective. This person doesn’t listen to the advice of the team, eve when they might tell them something that might help a process run smoother. The next type of poor leader I would call the abuser of power. They are people that get into leadership and use their power for personal gain. An example of this would be a boss that leaves early for the day all the time to take care of their personal business. The next type of poor leader is a leader that can’t be trusted. This is someone who employees don’t trust with information that can be used against them. When they have a problem the don’t feel comfortable talking to their leader in fear that they will tell someone else or use it against them in the future. They can’t tell them how they really feel. There is always going to be corrupt people in the world but when a leader is corrupt in any organization it breeds more corruption. There are some leaders that are down rite evil. They may have had a hard life and just don’t like to see others happy so they always find a reason to be upset and not satisfied with anything that is done within their department. Manager that operate in this manor normally don’t last long in leadership because there would make their employees unhappy and we all know that an unhappy employee won’t be a good producer. There ways to improve a bad boss if they want to improve. One way to do that is to make sure that middle level supervisors and managers have the support they need from upper management. I believe that leadership training should be an ongoing thing. I feel that in every organization there should be a hotline that employees can call to give feedback on how they feel their leadership is going. Once that feedback is collected it should be used as a training tool. Listening to the employees and responding to their needs and wants is in my opinion, the most effective way to go. During the hiring process employers should make sure that the leader they has the characteristic of a good leader. No one wants a bossy dictator as a boss. I have been in the workforce for over ten years and in that time I have had more good managers than bad. I think that is because it all comes down to respecting a person and there position. There have been times when my coworker s has come to me complaining about our boss. What I have found is that most of the time it not the boss at all it’s the employee view of their boss. Most people who don’t like their boss don’t like their job. I believe that if a person doesn’t like their job there is a likely chance they don’t like their boss because that the boss is only there to enforce the rules and doesn’t care employee’s personal feeling. In some cases they are right. The next time you are trying to determine if you have a good leader look for key traits. One key trait of a good boss is vision. Do they have a vision for the department that they will be managing? What is that vision? Does the vision line up with the companies mission statement? Ask yourself these questions. The next trait you should look for is wisdom. What useful knowledge can this person bring to the organization? Good leaders are strategic, wise and perceptive. The next question you should ask yourself about your boss is if he people are they passionate about what he or she does? Good bosses are very passionate people. They operate with such a high level of passion that they get consumed in it. They take action! Good leaders are determined to attain their goals. They know that the journey reaching their destination can be filled with problems. However, they are persistent people and are willing to put in the long hour and hard work to ensure that th eir goal is meet. Good leaders mean what they say. They have integrity. Theyre individuals who keep their guarantees and they do not play the old political games that plenty of others do. People find them reliable and as such are dedicated to them. Not all good bosses carry every single trait. They might be strong in some areas and lacking in others. But just remember, it isnt about being perfect, but understanding that bosses are human too; no one is perfect. Make an effort to try and get to know your boss and be a little more understanding about what they might be challenged with in their day to day duties.

Medieval Torture Essay -- essays research papers

Torture (Latin torquere, â€Å"to twist†), in law, infliction of severe bodily pain either as punishment, or to compel a person to confess to a crime, or to give evidence in a judicial proceeding. Among primitive peoples, torture has been used as a means of ordeal and to punish captured enemies. Examination by torture, often called the â€Å"question,† has been used in many countries as a judicial method. It involves using instruments to extort evidence from unwilling witnesses. In ancient Athens, slaves were always examined by torture, and for this reason their evidence was apparently considered more valuable than that of freemen. A free Athenian could not be examined by this method, but torture may have been used occasionally in executing criminals. Under the Roman Republic only slaves could be legally tortured, and as a general rule, they could not be tortured to establish the guilt of their master. Under the Roman Empire, however, by the order of the emperor, torture was frequently inflicted even on freemen to obtain evidence of the crime of laesa majestas (â€Å"injured majesty,† or crime against a sovereign power). The statesman Cicero and other enlightened Romans condemned the use of torture. Until the 13th century torture was apparently not sanctioned by the canon law of the Christian church; about that time, however, the Roman treason law began to be adapted to heresy as crimen laesae majestatis Divinae (â€Å"crime of injury to Divine majesty†). Soon after the Inquisition was instituted, Pope Innocent IV, influenced by the revival of Roman law, issued a decree (in 1252) that called on civil magistrates to have persons accused of heresy tortured to elicit confessions against themselves and others. This was probably the earliest instance of ecclesiastical sanction of this mode of examination. During the Middle Ages the influence of the Roman Catholic Church contributed to the adoption of torture by civil tribunals. The Italian municipalities adopted torture early, but it did not appear in other European countries until France legalized its use in the 13th century. Ultimately, torture became part of the legal system of every European nation except Sweden and England. Although torture was never recognized in the common law of England, it was practiced by exercise of the royal prerogative. In the American colonies torture was illegal; the few instances of its use were in ... ...nclose the head. Some resembled iron masks, with holes for mouth, nose and eyes. The victims mouth was clamped shut by an iron band passing under the chin and a flat piece of iron projected inside her mouth. This mouthpiece was sometimes armed with a short spike. The whole contraption was fastened round the neck with a heavy padlock. Branks were not only used to punish nagging wives. Any women found guilty of malicious gossip and slander, abusive language or breaches of the peace were silenced in this way. The branks were also padlocked on women convicted of witchcraft and condemned to die at the stake - but for a different reason. They prevented the unfortunate creatures for screaming horrible curses on their tormentors. References Medieval Punishment - Torture and Executions in Europe - 1100-1600 -- Cy Stapleton Rack, Rope and Red-Hot Pincers: A History of Torture and Its Instruments by Geoffrey Abbott Reissue edition (July 1995) Trafalgar Square The Book of Execution: An Encyclopedia of Methods of Judicial Execution by Geoffrey Abbott Reprint edition (August 1995) Trafalgar Square Torture by Edward Peters Expanded edition (December 1996) University of Pennsylvania Press