NE102 Final Lab Write-Up

Next the secondary antibodies were diluted using 5 ml

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Next, the secondary antibodies were diluted using 5 mL of blocking buffer. The blocking buffer was added to 1.5 µL of Gt α-Rb IgG and 0.5 µL of Gt α-Ms IgG each. After thirty minutes passed, we removed the primary antibody solution and washed our blots with TBST. Each membrane was washed with 10-15 mL for approximately three seconds. We repeated this step two more times. On the last wash, we placed the blots on a shaker for five minutes. This was also repeated two more times. Following the TBST wash, we incubated the membranes with the secondary antibody solutions for another thirty minutes. They were washed with TBST once again, and were now ready for visualization. This entire process was repeated for the PC12 cells that were transfected with the vector and the ZnEgr1. Except, for the primary antibody solution, we used 5 µL of Rb α-NFT L and 0.5 µL of Ms α- β-actin. For the secondary antibodies, we used 2.5 µL of Gt α-Rb IgG and 1 µL of Gt α-Ms IgG. Immunocytochemistry. Before ICC was performed, two chamber slides were plated with 200,000 cells each. For the cells with the Egr1 protein, the left chamber was left untreated with neuronal growth factor (NGF) and the right chamber, labeled “NGF-1h” was treated one hour before being used in the experiment. For the cells with the NFT L protein, the left chamber was again left untreated with NGF and the right chamber was treated for two days before being used in the experiment. We first pipetted 1 mL of 4% formaldehyde
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into each chamber of cells and incubated for ten minutes. Then we removed the formaldehyde and added 1 mL of PBS into each chamber and incubated for five minutes. We washed the cells two more times with PBS. Then 1 mL of 0.5% Triton X-100 was added to the cells and incubated for ten minutes. After that, we did a PBS wash three more times. The 500 µL of 5% NGS was added to each chamber and incubated for an hour. Similar to immunoblotting, we diluted the primary antibodies but using 1.1 µL of Rb α-NFT L and 11 µL of Rb α-Egr1. After the incubation, the primary antibodies were added to the chambers. The secondary antibody we used was Gt α-Rb-Alexa 488. Before adding them to the chamber, we diluted them with 2.2 mL of 5% NGS, using 22 µL of the secondary antibody. We washed the cells with PBS and then added 1000 µL of the secondary antibody solution and incubated for a hour. We prepared glass slides for visualization. Restriction Digests To create the restriction digests used in the expression plasmid for Ras and GFP We added specific volumes of deionized water, reaction buffer and the digests to the tubes as depicted in Table 1 below. Each of the tubes were flicked to ensure proper mixing of the various solutions.
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GFP/H GFP/HS Ras/B Ras/BE dH 2 O 42.5 µL 42 µL 42.5 µL 42 µL Reaction Buffer 5.5 µL 5.5 µL 5.5 µL 5.5 µL pGFP 1.5 µL 1.5 µL ----- ----- pRas-G12V ----- ----- 1.5 µL 1.5 µL Hpa 1 0.5 µL 0.5 µL ----- ----- Sma 1 ----- 0.5 µL ----- ----- BamH 1 ----- ----- 0.5 µL 0.5 µL EcoR 1 ----- ----- ----- 0.5 µL Total volume 50 µL 50 µL 50 µL 50 µL Table 1. Specific volumes of deionized water, reaction buffer and digests that were put in the 1.5 mL tube to create the different restriction digests.
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Next the secondary antibodies were diluted using 5 mL of...

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