Notes 20100504 Catherine Defense
From SnOwy - Ed's Wiki Notebook
Comparative Proteomics
- Catherine Wei
- Tag: Chinese Hamster Ovarian Cells, Intrinsic Apoptosis Pathway, Proteome; no artificial induction
- Intrinsic pathway to apoptosis: Mitochondrial membrane potential
- Apoptosome a complex that activates Caspase 9 -- leads to executioner caspases etc.,
- Intrinsic and extrinsic pathways linked at mitochondrion step
- Exponential, stationary and death phases
- Day 2.5 -- exponential phase (healthy)
- Apoptosis starts at day 4.5 (the tail of exponential phase -- early stationary phase, apoptosis starts)
- Accumulation of dead cells occurs at day 5.5 -- plasmalemmae start to leak
- Day 8.5 -- death phase
- Additional proteomic study guided by the above observed time points
- Flow cytometry cell subtypes -- compare levels of caspase 8, caspase 9
- 12 DIGE gels result from comparison of three pairs of time points
- Three heatshock proteins were down regulated (involved with cell survival)
- Cleavage of cytoskeletal proteins
- induce ER chaperones (=> Unfolded Protein Response (UPR) onset)
- upregulation of six glycolysis proteins (enables completion of apoptosis in low energy environment)
Brain Storms
- Monoclonal antibody production
- DIGE: Differential in gel electrophoresis
- MS: Mass spectrometry (protein sequence determined, used to search database)
- Flow cytometry
- Propidium Iodide (PI) - Membrane permeability, viability stain
- Protein major functions (in this study): Cytoskeleton, Chaperone, Metabolism
- Cytoskeleton: Vimetin, α-tubulin, β-tubulin, actin(β/γ)
- Endoplasmic reticulum chaperone systems -- facilitates protein folding; could have been triggered by ER stresses.