In lab for this semester, we isolated LDH from beef heart homogenate. However, I

Question

In lab for this semester, we isolated LDH from beef heart homogenate. However, I still don't have any idea on what we should have seen with regards to activity in each protein assay. This is a summary of what we did for each lab and the tables that follow. I really just need help understanding which of the data in my tables are right and which should have different numbers (some assays shouldn't have activity).

Enzyme Purification: Ammonium Sulfate. Beef heart homogenate was obtained and centrifuged at 20000 x g for 15 minutes (consistent with all other centrifugations). Ground ammonium sulfate was added at .230 g per milliliter of supernatant creating a 40% ammonium sulfate cut. The 40% cut pellet was resolubilized with 2 mL of phosphate buffer and the 40% supernatant centrifuged. 166 g of ground ammonium sulfate was added per milliliter of supernatant, creating the 65% cut. The solution was centrifuged. The supernatant was collected, and the pellet was resuspended in 5 mL of phosphate buffer and then placed into a Slide-a-Lyzer and dialyzed in .03 M bicine.

          Analysis of Fractions. A reaction cocktail was created which contained 1.9 mL CAPS buffer (0.14M, pH 10), 0.5 mL NAD+ (6mM), and 0.5 mL of lactate (0.15M, pH 10) and added to each fraction so that each assay had a total volume of 2.9 mL. 10µL of enzyme mixed with 90 µL of water was added to the assay. The Spec 20 was then set to 340 nm and used to obtain the change in absorbency of each assay per minute.

          Enzyme Purification: Column Chromatography. The Q-sepharose was ran with 0mM NaCl buffer (flow through fraction). A step gradient was then ran with 20 mL of NaCl buffer that increased by .2M every time the buffer was added, starting with .2M. The Spec 20 was set up at 280 nm to obtain absorbencies of each fraction. The pooled fraction is created by adding samples with the most activity.

          Protein Concentration Analysis. 5 dilute samples of 1.5 mL of varying BSA concentrations were made. 1.5 mL Coomassie reagent was then added to each sample. The Spec 20 was set at 595 nm to obtain absorbencies of each sample. This procedure was then repeated with the protein samples.

          Enzyme Kinetics. The reaction cocktail was made and included a volume of LDH in which the change in absorbency per minute was between .15 and .25. Varying amounts of NAD+ was then added to 2.15 mL CAPS buffer, .5 mL lactate, and .10 mL (water + enzyme). The Spec 20 was then set to 340 nm and absorbencies were recorded for each mixture with varying NAD+ amounts.

          SDS-PAGE and Western Blots. 2 identical gels were ran including each protein assay: 25 µl pooled protein, 10 µl dialysis sample, 5 µl each pellet and supernatant, 1 µl LDH from Sigma-Aldrich, and 10 µl of the protein standard. Water was added to each assay to make each assay 27.5 µl and then 12.5 µl of the 4x sample loading buffer was added to make each sample 40 µl total. These samples were then loaded into the gel and ran. The gel was then washed and then mixed with GelCode Blue stain reagent followed by another wash. The gel was then transferred into a sandwich including a membrane that was washed with 100% methanol for 15 seconds. Filter paper and sponges were put on top of and under them, respectively. The sandwich was then ran at 40V for 1 hour. The blot was removed and placed in 30 mL 2% milkfat TBST buffer followed by the cold room for 1 week. A Western blot was then ran with the blot using TBST, 1° antibody solution (1:5000 dilution), and 2° antibody solution (1:10000 dilution). An Alkaline phosphatase tagged enzyme was used as the color development agent as well as the Western Blue stabilized substrate from Promega.

These are the absorbancies calculated for the first lab. I'm not sure which fractions should have activity and which ones shouldn't.

Here is a breakdown of everything. Again, I'm not sure what's right and what's wrong. if you can help me out, it would be greatly appreciated.

Time (min) 20000 x g supernatant 40% supernatant 40% pellet 65% supernatant 65% pellet 65% dialysis pooled 0.000 0.200 0.090 0.079 0.161 0.201 0.123 0.047 0.167 0.267 0.156 0.086 0.138 0.430 0.225 0.073 0.333 0.321 0.201 0.082 0.122 0.544 0.327 0.094 0.500 0.362 0.243 0.079 0.104 0.665 0.387 0.114 0.667 0.399 0.301 0.076 0.091 0.772 0.452 0.135 0.833 0.437 0.320 0.073 0.085 0.845 0.514 0.158 1.000 0.470 0.357 0.071 0.085 0.934 0.573 0.175 1.167 0.501 0.392 0.077 0.088 1.040 0.621 0.194 1.333 0.534 0.422 0.082 0.090 1.075 0.671 0.217 1.500 0.564 0.451 0.084 0.092 1.144 0.720 0.233 1.667 0.591 0.485 0.084 0.094 1.179 0.770 0.251 1.833 0.618 0.510 0.085 0.097 1.234 0.798 0.265 2.000 0.642 0.541 0.085 0.098 1.281 0.839 0.281 2.167 0.669 0.567 1.351 0.881 0.298 2.333 0.690 0.590 1.398 0.913 0.314 2.500 0.709 0.616 1.403 0.945 0.328

Explanation / Answer

The values given in the table are correct.

20000g supernatant should have activity due to the presence of LDH enzyme.

The 40% supernatant will have the enzyme as the amount of ammonium sulphate added was less due to which the the protein cannot be pelleted out. The 40% pellet will not have any enzyme activity.

The 65% pellet will have the enzyme activity the amount of ammonium sulphate added will precipitate the enzyme.

The dialysed lysate will have more enzyme activity compared to 65% pellet due to the concentration of the enzyme.

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