Total protein synthesis was measured as previously described (25 (link)). Briefly, wild-type (eIF2α-WT) or S51A mutant eIF2α (eIF2α-S51A) stable transfected cells were seeded in six-well plates, serum starved for 16 h and then deprived of methionine and cysteine for an additional 2 h using DMEM without the above amino acids (Gibco). Cells were then treated with 10% serum containing 10 μCi ml-1 of 35S-Met/Cys (Perkin Elmer). Cells were lysed in RIPA buffer and protein concentration was determined by bicinchoninic acid assay (Pierce). Equal amounts of protein were separated by SDS/PAGE or an aliquot of lysate was trichloroacetic acid-precipitated and counted in a scintillation counter (Beckman Coulter).
Protocol detail
Measuring Cellular Protein Synthesis
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Amino acids
Assay
Bicinchoninic acid
Buffer
Cells
Methionine
Protein
Protein synthesis
Ripa
Scintillation counter
Sds page
Serum
Trichloroacetic acid
Corresponding Organization : City University of New York
Other organizations : Brigham and Women's Hospital, Harvard University
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Variable analysis
independent variables
- EIF2α genotype (wild-type vs. S51A mutant)
- Total protein synthesis
- Seeding cells in six-well plates
- Serum starvation for 16 h
- Methionine and cysteine deprivation for 2 h
- Treating cells with 10% serum containing 10 μCi ml^-1 of ^35S-Met/Cys
- Positive control: wild-type (eIF2α-WT) cells
- Negative control: S51A mutant eIF2α (eIF2α-S51A) cells
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