Cell Signaling Technology
Phospho-S6 Ribosomal Protein (Ser235/236)
4858
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The general aim of this project was to assess protein synthesis in a variety of treatment groups using ribosomal protein s6 (rps6), a downstream component of the MTORC1 pathway. To do this we compared phosphorylated rps6 to total using western blotting.
Western Blot
Total cell lysates of H4IIE rat hepatoma cells
1:2000 antibody, 0.1% tween, in 1:3 li-cor odyssey block: TBS overnight at 4 degrees C.
Li-Cor Odyssey Block for 1 hour.
1:15,000 Li-Cor IRDye 680RD, 0.1% tween, 0.02% SDS, in 1:3 odyssey block: TBS at room temperature for 1 hour
NA
Li-Cor Odyssey infrared Imaging system
There was a large distinction between samples that were phosphorylated (indicating active protein synthesis) and samples that were not phosphorylated (indicating halted protein synthesis). These results were consistent with another experiment performed to observe total protein synthesis.
None
Worked the first time using our normal protocol for detecting antibodies
Nothing
Had no issues in detecting rps6 and phospho-rps6 in this rat hepatoma cell line.