Determining the Amount of Protein in Each Homogenate: Hepa-1 cells were infected with many EYFP expression plasmids that had be previously homogenized and denatured in Laemmli buffer. The protein samples were diluted to a concentration of 1μg/μl. The different tubes are labeled as follows: GOL for golgi, TUB for tubulin, ACT for actin, MIT for mitochondria, and HEP for non-transfected Hepa-1 cells.
SDS-PAGE: A gel was obtained, then while wearing gloves the package was cut open and gel was removed from the cassette. Before putting the gel in the apparatus, the tape on the bottom that is covering the slot was removed. The chambers were filled up with 1X running buffer. Each of the samples were heated in the heating block for about a minute and …show more content…
The nitrocellulose piece was first submerged in nanopure water. Then, the blot sandwich was assembled in a tray of transfer buffer. One side of the transfer clamp was placed on the bottom of the tray and filled with transfer buffer. One Scotchbrite pad was submerged onto the bottom of the transfer tray. A piece of blotting paper was submerged onto the pad. The gel was then removed from the cassette and the top left corner was clipped. The gel was laid on top of blotting paper with the clipped end on the top right. Then, the nitrocellulose membrane was removed from the water and carefully placed on top of the gel without trapping any bubbles between them. The sandwich was finished by adding the second piece of blotting paper and the second Scotchbrite pad. Keeping it submerged, the sandwich was closed. Finally, the sandwich was quickly transferred to the gel box, then filled with buffer to the top of the sandwich. The transfer was ran at 100 mA for one …show more content…
Ponceau S dye was added to cover the blot and swirled for a few seconds. Then, the dye was dumped back into the bottle and the blot was rinsed with distilled water. Once most of the dye was washed off, the blot was blocked with 5% dry milk solution. The blot was incubated in the solution for 30 minutes and then washed several times with PBS solution. The blot was then placed on a Whatmans paper and stored.
Western Blot Staining: The blot was removed from the saran wrap and carefully submerged into a staining tray with 15 ml of TBS and left to rest for a few minutes. The primary antibody solution with a dilution factor of 1:1000 was obtained. The TBS was then dumped down the drain and the primary antibody solution was added to the blot. The tray was rocked by hand gently to cover the blot. The tray was covered with saran wrap in order to prevent evaporation, then put on the rocking platform for 45
SDS-PAGE: A gel was obtained, then while wearing gloves the package was cut open and gel was removed from the cassette. Before putting the gel in the apparatus, the tape on the bottom that is covering the slot was removed. The chambers were filled up with 1X running buffer. Each of the samples were heated in the heating block for about a minute and …show more content…
The nitrocellulose piece was first submerged in nanopure water. Then, the blot sandwich was assembled in a tray of transfer buffer. One side of the transfer clamp was placed on the bottom of the tray and filled with transfer buffer. One Scotchbrite pad was submerged onto the bottom of the transfer tray. A piece of blotting paper was submerged onto the pad. The gel was then removed from the cassette and the top left corner was clipped. The gel was laid on top of blotting paper with the clipped end on the top right. Then, the nitrocellulose membrane was removed from the water and carefully placed on top of the gel without trapping any bubbles between them. The sandwich was finished by adding the second piece of blotting paper and the second Scotchbrite pad. Keeping it submerged, the sandwich was closed. Finally, the sandwich was quickly transferred to the gel box, then filled with buffer to the top of the sandwich. The transfer was ran at 100 mA for one …show more content…
Ponceau S dye was added to cover the blot and swirled for a few seconds. Then, the dye was dumped back into the bottle and the blot was rinsed with distilled water. Once most of the dye was washed off, the blot was blocked with 5% dry milk solution. The blot was incubated in the solution for 30 minutes and then washed several times with PBS solution. The blot was then placed on a Whatmans paper and stored.
Western Blot Staining: The blot was removed from the saran wrap and carefully submerged into a staining tray with 15 ml of TBS and left to rest for a few minutes. The primary antibody solution with a dilution factor of 1:1000 was obtained. The TBS was then dumped down the drain and the primary antibody solution was added to the blot. The tray was rocked by hand gently to cover the blot. The tray was covered with saran wrap in order to prevent evaporation, then put on the rocking platform for 45