Experimental Method:
Extracting LDH. The LDH used was extracted from bovine skeletal muscle tissue. In the cold room, a mass of 20 grams of muscle tissue was obtained. It was placed in a blender along with ice cold buffer, 50 mM sodium phosphate and 10 mM EDTA at pH 7.5. The mixture was then blended and homogenized in order to break open the cells; during this time the temperature was checked periodically to make sure it stays below 20 C. This was done until the solution was homogeneous. The solution was then transferred to a centrifuge tube and centrifuged. The supernatant was aliquoted and stored in a -20 C for use in future experiments.
Enzymatic Assay of LDH by Spectrophotometry. One tube of tissue extract was thawed out on ice and a …show more content…
To make a 12.5% resolving gel, a mixture of 30% acrylamide, 0.8% bisacrylamide, 4X Tris HCl buffer with pH 8.8, DI water, 10% ammonium persulfate, and TEMED. The solution was poured into a gel caster, ethanol was added on top, and it was left alone to polymerize. A 4.5% SDS stacking gel was then prepared by mixing: 30% acrylamide, 0.8% bisacrylamide, 4X Tris HCl buffer with SDS pH 6.8, DI water, 0.25% bromophenol blue, 10% ammonium persulfate, and TEMED. The gel was left to polymerize and the running buffer was prepared similar to the previous step. Following this the protein samples were prepared by calculating the volume of protein needed to add to each sample, by taking the amount of protein given and dividing by the protein concentration found on the table completed in experiment seven (Table 1). The samples were mixed in an Eppendorf tube, heated for 60s, and centrifuged for 5s. While the samples were being prepared, the electrophoresis chamber was prepped in the lab and the running buffer was poured into the chamber. The samples were then loaded into the wells, the power supply was connected, and ran for 45 minutes at 250V. The power was shut off when the dye was close to the end of the gel. the gel was placed into a tube of Simply Blue Coomassie stain mixed with 20% NaCl and microwaved in order for the bands to develop. It was then distained and a picture of the gel was
Extracting LDH. The LDH used was extracted from bovine skeletal muscle tissue. In the cold room, a mass of 20 grams of muscle tissue was obtained. It was placed in a blender along with ice cold buffer, 50 mM sodium phosphate and 10 mM EDTA at pH 7.5. The mixture was then blended and homogenized in order to break open the cells; during this time the temperature was checked periodically to make sure it stays below 20 C. This was done until the solution was homogeneous. The solution was then transferred to a centrifuge tube and centrifuged. The supernatant was aliquoted and stored in a -20 C for use in future experiments.
Enzymatic Assay of LDH by Spectrophotometry. One tube of tissue extract was thawed out on ice and a …show more content…
To make a 12.5% resolving gel, a mixture of 30% acrylamide, 0.8% bisacrylamide, 4X Tris HCl buffer with pH 8.8, DI water, 10% ammonium persulfate, and TEMED. The solution was poured into a gel caster, ethanol was added on top, and it was left alone to polymerize. A 4.5% SDS stacking gel was then prepared by mixing: 30% acrylamide, 0.8% bisacrylamide, 4X Tris HCl buffer with SDS pH 6.8, DI water, 0.25% bromophenol blue, 10% ammonium persulfate, and TEMED. The gel was left to polymerize and the running buffer was prepared similar to the previous step. Following this the protein samples were prepared by calculating the volume of protein needed to add to each sample, by taking the amount of protein given and dividing by the protein concentration found on the table completed in experiment seven (Table 1). The samples were mixed in an Eppendorf tube, heated for 60s, and centrifuged for 5s. While the samples were being prepared, the electrophoresis chamber was prepped in the lab and the running buffer was poured into the chamber. The samples were then loaded into the wells, the power supply was connected, and ran for 45 minutes at 250V. The power was shut off when the dye was close to the end of the gel. the gel was placed into a tube of Simply Blue Coomassie stain mixed with 20% NaCl and microwaved in order for the bands to develop. It was then distained and a picture of the gel was