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 Mystery of Life and Death Essays

The Mystery of Life and Death Essays The Mystery of Life and Death Essay The Mystery of Life and Death Essay The Mystery of Life and Death In the 1995 movie Breather, William Wallace presents lifes uncertainties and how we should live each day when he affirms, Every man dies. Not every man really lives. Sharon Oldies poem, Summer Solstice, New York City, is an ideal representation of this quotation and the questions that we have about how to live our day-to-day lives. This brief poem is about a man who is standing on a rooftop contemplating suicide and the New York City policemen who are attempting to save his life. At first, a brief summary is all that the reader sees in the story, but upon peer analysis, the fragility of life shines through. With each detail, or lack thereof, Olds reveals her views on the uncertainties of this life. The Summer Solstice is known as the longest day of the year, and in placing the setting on the Summer Solstice, Olds presents the reader with a lengthy period of time. In the first line, when Olds says that he could not stand it (1) anymore, it presents the concept of a long day, or in the case of the man, a long life and a long time coming. We do not know what has been happening in the mans life, but whatever it is has led him to desire to commit suicide. The man on the rooftops identity remains a complete mystery throughout the entirety of the poem. There is no physical description of the man; the descriptions in the story are of his actions. The lack of description of the man allows readers to apply the mans actions and the story to themselves, rather than merely reading a poem and remaining on the outside. We barely know anything of the mans story except that by the end of the longest day of the year he could not stand it (1). The question remains: What could the man not stand anymore? Some of us may have felt his pain before and are able to fully relate to feeling suicidal, while others understand the concept of being increasingly upset with someone or something. In her word choice, Olds invites the reader to identify with the man and his struggle, whatever our identification with the matter may be. As the man is standing on the roof, he is poised between life and his immediate death. When the man reaches the edge of the roof, without hesitation, he putts] one leg over the complex green tin cornice (4). Hesitation begins to rush over him once he puts his leg over the edge. As the mans leg [hangs] over the lip of the next oral (18), the readers are given strong imagery of how quickly ones life can change. One half of his body is pointed toward life, while the other half of his body is on the side of death; any quick movement in each direction could determine the mans life. This description compares to our lives directly; any action that we take could determine our lives forever. This mystery of death is what attracts all of the people below; they are prepared for the worst. The people [gather] in the street, silent (19) as the cops came in their suits blue-gray (7); they watch as the hairy net with its implacable grid was unfolded near the curb and spread out and stretched as the sheet is prepared to receive at birth (20-22). The scene that is described here is one of waiting on the possibility of death. The policemen are working to prevent what could happen, while the bystanders are awaiting a finale, watching the man balance they are merely interested in the aspect of danger and the fear of someone dangling at such a high altitude with no obvious desire to walk away from the edge. The people standing down below are attempting to place themselves into the mind of the an up on the rooftop understand why he is standing on the edge of the building and why he has not yet Jumped. We are left guessing as to what brings the man to spare his life. Was he actually suicidal? Did the policemen convince him not to take his own life? We are never told the reasons for the mans not Jumping from the building; we only know that everything stopped as his body Jerked and he stepped down from the parapet and went toward them (27-29). We are presented with so few details so that we are able to compare the actions of the man to our own previous experiences or actions. The Nans actions were Just as possible as those which he did not take. The outcome could have been different, and the purpose of the story would remain the same. Life is uncertain, and the line between life and death is extremely thin. As the Summer Solstice marked the official beginning of summer, ancient civilizations used this opportunity to celebrate by throwing festivals and enjoying the day together (Dunlap Patch). At these celebrations, participants would have a bonfire to celebrate the sun and its rays and power (Dunlap Patch). These celebrations connect directly with the ending of the poem, when the policemen have adhered around the man and continue to smoke cigarettes that burned like the tiny campfires we lit at night back at the beginning of the world (38-40). Though the ending of the poem can be interpreted in various ways, the relationship between Summer Solstice, New York City and the holiday for which the poem is named is clear to the reader after further analysis. The comparison between life and death has been depicted in various ways throughout literature and all areas of the arts because audiences are drawn to the mysteries behind the characters. We long for the unknown, as it opens a door to our imagination and creativity. We already have an ending to the story in our heads before the characters can present us with a resolution. By using omission of details, Olds allows us to interpret the poem individually to determine our own views of the world and of our lives. Breather. Dir. Mel Gibson. Peer. Mel Gibson. Paramount Pictures, 1995. Film. Cassia, Ron. Summer Solstice: A Brief History of a Midsummers Traditions. Dunlap Patch. N. P. , 21 Jan. 2011. Web. 24 Gag. 2013.. Olds, Sharon. Summer Solstice, New York City. Making Literature Matter. 5th deed. Deed. John Schlitz and John Clifford. Boston: Bedford, 2012. 21-22.

Amercian History Essay Example | Topics and Well Written Essays - 750 words - 1

Amercian History - Essay Example Although the colonies were self-governed from thirty years earlier, the straining relationship with the British crown became more acute with the passage of each new tax law. The series of tax laws, including the Sugar Act, the Stamp Act and the Currency Act aggravated the grievances of an already over-taxed colonial population. Almost all trade-related shipments to and from the Eastern American coast was deemed taxable. While some of the taxes were reasonable, others were plainly unjust, given that the colonists were not granted representation in the affairs of the Crown. The British Crown reasoned that such taxations were necessary to keep up its expansive and expensive naval operations. But its use of authoritarian methods in enforcing tax laws proved to be the decisive spur for American Revolution. For example, when some of the governing officers in the colonies expressed their solidarity with fellow colonists, the British Crown took a hasty decision to dismiss and replace all dis loyal officers and took more stringent measures to enforce taxation laws. This move further alienated the colonists and primed their thoughts for independence. Studying the American declaration of independence in retrospect, we see that the British rule, by its obstinate, inconsiderate and high-handed approach to dealing with colonial affairs, had triggered the revolt. By 1770 the movement for independence had gathered substantial following, making it a full blown war against Britain. Colonists gathered in huge numbers and participated in protestations. They applied paints on their faces, wore Mohawks as a mark of identifying with America, while simultaneously distinguishing themselves from British troops. It was at this juncture that the famous Boston Tea Party happened. Acting in disobedience to the orders given from England, the colonists marched down onto the ships that sat in the bay that contained crates of tea. After