Ldh Purification Lab Report

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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.

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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.

Affinity Chromatography Lab Report

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 NADIA can be determined spectrophotometer’s 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 spectrophotometer’s 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.