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 Perversity Of The Congo Essays - Congo Free State, Free Essays

The Perversity Of The Congo Essays - Congo Free State, Free Essays The Perversity Of The Congo In the novel Heart of Darkness by Joseph Conrad one of the major themes is the perversity of the Congo. What is good and evil in the European world becomes distorted and hazy in the heart of Africa. To the outside world white is good and black is evil; it is as simple as that. This philosophy is embodied in Marlows aunt, who believes that his job is to bring light into the land of darkness and to enlighten the savages. This idea, however, becomes corrupted when white objects symbolize suffering and greed instead of good, and light images hide the presence of darkness. Symbols such as, a white rag, white imperialists and ivory, no longer represent the good will of the imperialists, on the other hand they represent the exploitation and chaos that the Europeans have brought to the Congo. The main character Marlow is faced with this confusion as he voyages through the jungle, and he must reevaluate his former opinions, which no longer hold true. The European philosophy is shown through the conversation that Marlow has with his aunt before commencing his adventure. According to her, his job seems clear: to bring civilization and light to the heart of darkness. Instead of focusing on the horrors of imperialism she is disillusioned to believe that it is all for the better. The Europeans, especially the British have no respect for other cultures or other ways of life, and they truly believe that they are helping the Africans. Not by choice but because of the white mans burden they feel the need to [wean] those ignorant millions from their horrid ways(28). To the outside this seems like an earnest motive; however, once inside Marlow begins to see new forms of corruption. Are the imperialists their to help, or are they there to make money to fulfill their greed? He begins to realize that it is not the black savages who represent evil, but rather the selfish whites. This corruption is further shown through the novel with symbols that reveal that perversity of the jungle. None of Marlows previous beliefs hold true in the Congo and he must reevaluate what is light and what is dark. He is confronted with the distortion of images and confusion at the first station. He sees a group of natives in the shade and immediately compares it to hell. As he states: Black shapes crouched, lay, sat between the trees, leaning against the trunks, clinging to the earth, half coming out, half effaced within the dim light, in all the attitudes of pain, abandonment, and despair(35). He notices one figure in particular, one with a white rag around his neck. Is it the natives who create this feeling of suffering or is it the whites? These people are in the shade because they have nothing to live for anymore. The imperialists have destroyed their way of life and now they are eagerly awaiting death. The corruption is not in the black boy, rather in the white rag. What it symbolizes is not clear. Marlow asks, Where did he get it? Was it a badge an ornament a charm a propitiatory actIt looked startling round his black neck, this bit of white thread from beyond the seas(35). Marlow does not know why exactly the boy is wearing the rag; however, he does know that the Europeans brought it - along with suffering and corruption. Rather than bringing light to the natives, they have brought nothing but pain and chaos. This confusion in appearances is show again with the alternative motives of the whites. They are not humanitarians helping a civilization out of good will. They are there out of greed and corruption. Without the presence of society, the inner core of humans is revealed and what is white on the outside is sometimes black on the inside. This reversal of appearances is displayed in all the imperialists that Marlow comes across. One is the manager at the first station. He gives the allusion of being a gentleman with his European clothing and manners, yet inside he is filled with crookedness. In order to maintain this image he must train a native to follow his