LDH Purification Table Analysis PDF

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LDH Purification Table Analysis Purification Intermediate

Relative activity (U/ml

Protein LDH Recovery by that Step (%)

Protein Concentrati on (mg/ml)

Total Protein (mg)

Specific Activity (U/mg)

Clarified Homogenate

10.45

710.6 (2000)

N/A

14.65

996.2 mg

0.7133 (2.01)

65% cut Pellet

245.2

1483.3

208.74 (74.17)

88.91

537.9

2.76

65% Cut Supernatant

0.7235

56.433

7.94 (2.82)

N/A

N/A

N/A

Affinity Purified LDH

0.643

5.144

0.7

3.617

28.936

0.1777

Size-Exclusion Purified LDH

12.058

72.348

42.12

6.737

40.422

1.7898

Figure 1: (Units KDa)

Total LDH Activity Units (U)

LDH Standard

Molecular weight marker

Size Exclusion

Affinity Purification

65% Cut Pellet

Clarified Homogenate

From Left to Right Q1) The Affinity purification technique lost the most LDH activity. This is according to the percent of the protein recovered at 0.7% and based on the relative activity 0.643 U/ml. When the SDS-Page gel is consulted (Figure 1 ) affinity purification had the lowest band for LDH present compared to size exclusion. While this step lost the most mass as proportion of total protein (mg), most of this eluted mass must have contained LDH activity units. This decrease in mass would thus explain the significant drop in relative activity and total activity from the 65% pellet to the affinity purified LDH. This drop could be in part due to several possible factors: a low Kd dissociation constant, cibacron Blue-agarose that was not packed tightly enough, or a lack of an NaCl gradient. A low Kd constant would mean that the ligand receptor complex would precipitate at a greater rate the more phosphate buffer added. This is shown by the equation Kd [L][R]/[LR]. More of the protein initially being washed would thus have LDH activity units, and this would not be present in the collected sample. The lack of an NaCl gradient would disassociate the LDH activity units too rapidly and prevent dissociations based on a general linear trend of affinity that could be quantified. While the unpacked cibacron Blue-agarose beads would allow for an easier disassociation of the LDH activity from the beads earlier on in the initial wash with phosphate buffer. Despite the experiment results that show affinity purification as the technique with the most lost LDH activity, it would have been more common for the results to show size exclusion chromatography having the largest mass eluted, and having a much lower relative activity. This would be especially true for the range measured in this lab’s size exclusion chrootagrapy since G100 was used. Since the fractionation range of G100 is 4000-150,000 and LDH is 146.4 kDa , LDH is likely lost with the larger molecules. There is also more likelihood for size exclusion chromatography to have errors in elution time, column length, and sample packing. The darker bands shown at different areas beside the LDH marker on the SDS-page gel (figure 1) for Size exclusion chromatography show that this technique should have the least specific type of purification for LDH activity. Despite this however, the experimental variations show that for these trials affinity chromatography had the least specific type of purification.

Q2) A more effective purification strategy for LDH can be created by roughly using a majority of the same lab procedures. The source material would still be homogenized to make a crude material, and then centrifuged at 20,000x. This would break down cell structure and precipitate unwanted protein and structures.The supernatant would then also be taken out to be used as

the clarified homogenate. However a change would be made with the addition of a protease inhibitor, which would stop the degradation of LDH while the clarified homogenate is stored for cooling. The prevention of LDH degradation would thus make the specific activity of the clarified homogenate and the percent recovery of the 65% cut pellet more accurate. This would especially help this lab report results since clarified homogenate total activity was 710.6 U and was less than the theoretical value 2000 U. The presence of LDH would also be viewable on the SDS-PAGE GEL at the 36 kDA marker. As more LDH is present the bands should be darker. The following procedure would continue to use ammonium sulfate to eliminate and precipitate contamination proteins from the clarified homogenate. Switching from a 40% to 50-55% cut however would filter out a greater amount of non-LDH protein and ensure more LDH present overall in the 65% cut pellet. This would be shown on the SDS PAGE gel with a darker band for the 65% cut pellet as opposed to the blotched bands shown in figure 1. At the same time this change in cut would serve to increase relative activity by increasing LDH present, decrease concentration through removal of inconsequential protein, and overall increase specific activity and percent recovery. To ensure that LDH is not lost in the 50%- 55% cut pellet, a spot test of the supernatant and resuspended pellet from the clarified homogenate at the 50-55% cut could be tested. This would allow a qualitative result of LDH activity at this step, that would determine whether adjustments to the cut need to be made. The 65% cut pellet could then be created and proceeded to be suspended in cold buffer. Since size exclusion chromatography has a higher risk in general than affinity chromatography to be contaminated, it is a good candidate for the first chromatography test. However the test would be more accurate if G150 sephadex is used rather than G100. Increasing the column length as well to 60cm could also increase accuracy. G150 would change fractionation to 5kda to 300kda which insures LDH at 146.4 kDA eludes towards the middle. The 30 cm column length increase serves to increase separation for LDH. This creates purer elution volumes through overall better separation. These changes would create darker LDH bands on the Size Exclusion chromatography band on the SDS-PAGE gel. In addition percent recovery, relative, and specific activity would also increase. Like the previous lab procedure, a spot test would be conducted to determine the fractions from the column to be used in the next step. After size exclusion, affinity chromatography would be used to complete the purification of LDH. However, unlike the previous lab procedure a gradient from 0.5 M NaCl to 3 M NaCl would prove more effective. This creates fractions with smaller amounts of more specific protein. In conclusion of this step, spectrophotometry would still be used to ensure protein elution and a proper baseline. The spot test would conclude the testing. These changes would create an Affinity purified LDH with a darker band than the one seen in figure 1. It would also serve to increase relative and specific activity as well as decrease contaminated protein and total protein concentration. Activity assays, and Bradford assays would still be run on all intermediates to determine LDH present .Another possible approach however to increase accuracy of LDH concentration and activity would be to eliminate the 65% cut pellet. The data shows that total, specific,and relative activity from the size exclusion and affinity purification steps showed little overall recovery and activity. Combining more fractions from the spot test of the affinity and size exclusion chromatography could generate a higher recovery of protein than the 65% cut pellet. Applying this quantity to Ion exchange chromatography then could improve the protein recovery and LDH activity. By applying this sample to a DEAE-Toyo-Pearl column, LDH could be recovered more accurately since this column separates molecules more

accurately on net charge. In addition, the addition of oxalate as an inhibitor would also act to increase LDH specificity . This step would overall would result in an increase in LDH activity and specificity measured....