NIH3T3 Cells Lab Report

The samples were boiled for 3 minutes and centrifuged for 20 seconds. A polyacrylamide gel was assembled and the samples were loaded into it. The gel ran for approximately 45 minutes at 160 V. A western blot analysis was done in order to examine the Erk1/2 phosphorylation of the NIH3T3 cells. Three pieces of Whatman paper were wet in transfer buffer and placed on the anode plate. A wet PVDF membrane was placed on top of the Whatman paper. The gel was removed from holder and the stacking gel was removed. Forceps were used to place the gel onto the wet membrane and another Whatman paper was placed on top of the. The cathode plate was placed and started the transfer. Then the membrane was placed in TBST containing 5% milk for 60 minute while shaking. The membrane was rinsed twice with TBST (no milk) and placed in the primary antibody. The membrane was washed three times for two minutes
The goal of this experiment was to use cell cultures to monito the growth of these cells when treated with different calf serum and FGF concentrations. SRB protein assay was conducted in order to observe the cell proliferation. Western blotting was used to look at the relationship between the serum and activation of the Erk ½ pathway along with protein concentration. Thus, there were two hypotheses being tested during this experiment. This first hypothesis states: If various concentrations of Fetal Bovine Serum were added to non-transformed and transformed cells, the non-transformed cell density would increase with the concentrations of these substances and the transformed cells would be unaffected. The second hypothesis being tested is that the protein concentration of the Ras-transformed cells should also remain unaffected by the addition of serums and growth factors. However, the non-transformed cells will again increase with the concentrations of these