Methods And Materials:
Cell Preparation. Four bovine ovaries were obtained from the slaughterhouse, their corpora lutea were extracted, and their luteal cells were prepared. The cells were incubated in T-75 flasks containing Ham’s F-12 medium with 5% fetal calf serum, insulin, transferrin (5 mg/ml), selenium (5 ng/ml), penicillin (100 U/ml), streptomycin (0.1 mg/ml), and amphotericin B ( 0.25 mg/ml) in an atmosphere of humidified 95% air and 5% CO2 at 37°C. At confluence, the cells either remained untreated as a control or treated with fish oil at 3×10-3 (vol/vol) for 48 hours.
Fractionation. After 48 hours, the supernaut of the T-75 flasks were removed and the cells were trypsinized to remove them from the flasks. The cells were then centrifuged to form a cell pellet. The pellet was then transferred to 0.66 mL 90% sucrose. 1M MES solution was added and the whole solution was homogenized to a final volume of 1.32 mL 45% sucrose solution at 0.5M MES and 250mM Na2CO3 concentrations. Additional layers of 1.32 mL 35% sucrose and 1.32 mL 5% sucrose were carefully added to the 45% sucrose solution to form distinct layers in 12mm × 75mm ultracentrifugation tubes. Each treatment was then ultracentrifuged for 24 hours at 250000 xg at 4°C utilizing a SW-60 Beckman rotor.
Protein Isolation. After removing the samples from the centrifuge, 400μL aliquots were removed starting from the top of the ultracentrifugation tube and placed in 10 new, marked 1.7 mL eppe tubes. A 200μLof aliquot was placed in the 10 new eppe tubes with the sample. Then 200μL of trichloro acetic acid (TCA) was added to each sample. the proteins were then isolated overnight at 4°C. The samples were then centrifuged at 20000 xg for 10 minutes at 4°C. After centrifugation, the samples were washed in ice cold acetone. Samples were then centrifuged and acetone washed again. After being washed, the samples were reconstituted in a 15μL 1x running buffer for western blot analysis.
Western Blot Analysis. If samples were left in the freezer overnight, place on heat plate for 6 minutes, lids closed. Centrifuge for 30 seconds and place in test tube rack. Prepare gels by running empty in buffer for 15 minutes at 140V. When the gels are ready, add 7μL red ladder and blue ladder starting on the right and working towards the left. Insert 15μL of samples 1 to 10 from right to left using a different pipet tip between each sample. Add 1x running buffer to the middle of the Cell, and add old running buffer or DI H2O to the outer part of the Cell. Run at approximately 145V for an hour and a half. While waiting, prepare the transfer papers and pads by placing 5 fiber pads and 2 filter papers for each gel you run in transfer buffer. At the end of gel electrophoresis, remove the gel(s) from their plastic casing and place in transfer buffer to clean off any extra gel. Place gel in methanol for 2-3 minutes and then in HOH for another 5 minutes. Then place the gel(s) in transfer buffer for 15 minutes. At the end of the 15 minutes, stack 2 fibre pads, 1 filter paper, the gel, another filter paper, and 3 more fibre pads. Place in the Cell. Fill the inner box with halfway with transfer buffer and the outer part of the box with old transfer buffer or DI H2O. Run for 1 hour at 20V. After one hour, wash the gel(s) in TBS-t 5 times, 3 minutes each on a tilt-table. After the fifth wash, block gel(s) with 5% milk for 1 hour. After the hour has ended, add 10 mL of milk with 20μL of primary antibody overnight in the cooler. In the morning, rinse 3 times in TBS-t for 5 minutes each. Add 10 mL of milk and 3μL of the second antibody and 1μL of HRP. Place on the tilt-table for an hour. When the hour is up, wash 3 times in TBS-t for 5 minutes each and take to the imager.
Western Blot Imaging. Place the transfer paper into a petri dish and pour chemifluorescence onto the transfer paper with the proteins on it. Place the petri dish directly under the camera and allow the camera to position correctly. Set the camera to take the amount of pictures you want in the amount of time you want. Celebrate, stare, or cry because there are three types of Westerns, and yours has to be one of them.
Fractionation. After 48 hours, the supernaut of the T-75 flasks were removed and the cells were trypsinized to remove them from the flasks. The cells were then centrifuged to form a cell pellet. The pellet was then transferred to 0.66 mL 90% sucrose. 1M MES solution was added and the whole solution was homogenized to a final volume of 1.32 mL 45% sucrose solution at 0.5M MES and 250mM Na2CO3 concentrations. Additional layers of 1.32 mL 35% sucrose and 1.32 mL 5% sucrose were carefully added to the 45% sucrose solution to form distinct layers in 12mm × 75mm ultracentrifugation tubes. Each treatment was then ultracentrifuged for 24 hours at 250000 xg at 4°C utilizing a SW-60 Beckman rotor.
Protein Isolation. After removing the samples from the centrifuge, 400μL aliquots were removed starting from the top of the ultracentrifugation tube and placed in 10 new, marked 1.7 mL eppe tubes. A 200μLof aliquot was placed in the 10 new eppe tubes with the sample. Then 200μL of trichloro acetic acid (TCA) was added to each sample. the proteins were then isolated overnight at 4°C. The samples were then centrifuged at 20000 xg for 10 minutes at 4°C. After centrifugation, the samples were washed in ice cold acetone. Samples were then centrifuged and acetone washed again. After being washed, the samples were reconstituted in a 15μL 1x running buffer for western blot analysis.
Western Blot Analysis. If samples were left in the freezer overnight, place on heat plate for 6 minutes, lids closed. Centrifuge for 30 seconds and place in test tube rack. Prepare gels by running empty in buffer for 15 minutes at 140V. When the gels are ready, add 7μL red ladder and blue ladder starting on the right and working towards the left. Insert 15μL of samples 1 to 10 from right to left using a different pipet tip between each sample. Add 1x running buffer to the middle of the Cell, and add old running buffer or DI H2O to the outer part of the Cell. Run at approximately 145V for an hour and a half. While waiting, prepare the transfer papers and pads by placing 5 fiber pads and 2 filter papers for each gel you run in transfer buffer. At the end of gel electrophoresis, remove the gel(s) from their plastic casing and place in transfer buffer to clean off any extra gel. Place gel in methanol for 2-3 minutes and then in HOH for another 5 minutes. Then place the gel(s) in transfer buffer for 15 minutes. At the end of the 15 minutes, stack 2 fibre pads, 1 filter paper, the gel, another filter paper, and 3 more fibre pads. Place in the Cell. Fill the inner box with halfway with transfer buffer and the outer part of the box with old transfer buffer or DI H2O. Run for 1 hour at 20V. After one hour, wash the gel(s) in TBS-t 5 times, 3 minutes each on a tilt-table. After the fifth wash, block gel(s) with 5% milk for 1 hour. After the hour has ended, add 10 mL of milk with 20μL of primary antibody overnight in the cooler. In the morning, rinse 3 times in TBS-t for 5 minutes each. Add 10 mL of milk and 3μL of the second antibody and 1μL of HRP. Place on the tilt-table for an hour. When the hour is up, wash 3 times in TBS-t for 5 minutes each and take to the imager.
Western Blot Imaging. Place the transfer paper into a petri dish and pour chemifluorescence onto the transfer paper with the proteins on it. Place the petri dish directly under the camera and allow the camera to position correctly. Set the camera to take the amount of pictures you want in the amount of time you want. Celebrate, stare, or cry because there are three types of Westerns, and yours has to be one of them.