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NE102 Final Lab Write-Up

Methods light microscopy we captured images of the

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Methods Light microscopy. We captured images of the PC12 cells that were either treated with NGF or left untreated. This was done at two different objectives: 20x and 40x. Fluorescence microscopy. A fluorescence microscope was used to capture images of cells that were used in ICC to visualize certain proteins. We used this microscope to obtain images of the localization of NFT L and Egr1 in differentiated cells. We also took images of cells that were transfected with pGFP or pGFP and pRas-G12V. SDS-PAGE and Immunoblotting. We first took the proteins out of the cells by creating cell lysates. In order to make the cell lysates, we pipetted PBS onto the cell dish. Then we poured out the PBS into a
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waste beaker and repeated this step again. After, we pipetted 200 µL of lysis buffer onto the cell culture and used a cell scraper to spread the lysis buffer. Using a P1000 pipetman, the lysates were transferred into 1.5 mL tubes. The tubes were placed on a boiling rack to be put in boiling water for five minutes. We centrifuged the tubes after the boiling period. This same method was used to create lyses of the GFP and GFP plus Ras-G12V transfected cells, and the vector stable and ZnErg1 stable cells. We loaded the samples into the electrophoresis, adding in 5 µL of protein standard each time. For the cell lysates of the Ras protein, we added in 15 µL of each lysate in the corresponding wells and, for the Egr1 cells, we added in 20 µL of each cell lysate into the wells. We ran the gel at 200V for approximately forty minutes. While the gels were running, we prepared three staining trays with 10-15 mL of TBS in them. When the gels were completed, we took them out of their plastic covers and cut them into equivalent blots. These were then transferred to a nitrocellulose membrane. After the gels were cut, they were placed on top of the nitrocellulose membrane and both of them were put in between filter paper. The electroblotter ran for a total of seven minutes. After, we cut the filter paper to obtain identical blots. They were placed in their respective staining trays of TBS. We rocked the trays for approximately five seconds to wash the membranes and then poured out the TBS. We repeated this step three more times. The blots were now ready for immunoblotting. This was repeated for both Ras transfected cells and the Egr1 transfected cells. For the immunoblot, we added different amounts of antibodies for each of the two different experiments. For the cells transfected with GFP or GFP plus Ras-G12V, we removed the TBS and pipetted 5 mL of blocking buffer on the membranes. They were put
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on the shaker for thirty minutes while the primary antibodies were diluted. We used 5 µL of Rb α-Ras and 0.5 µL of Ms α- β-actin and diluted them with 5 mL of blocking buffer. Following the incubation, we removed the blocking buffer and poured the primary antibody solutions onto the appropriate blots for another thirty-minute incubation period.
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