Tour-de-Force

Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007Mitotic Shake-offMitotic shake-off is a procedure in cell biology used to synchronize cells. Cells have the natural tendencyto round up during mitosis and become less attached to the rest of the cell culture. By gently shaking theculture, the mitotic cells can be removed from the cell culture. Since cells that are not in mitosis are morefirmly attached to the cell culture, the suspension will contain cells in mitosis. To increase this number,cells can be first treated with a drug, for example colcemid, that blocks mitosis during metaphase.Mitotracker Green FMMitotracker Green FM is a dye directed towards mitochondria regardless of their mitochondrial membranepotential. It will appear as a green-fluorescent dye and can be used to stain living cells as well as fixedcells. However, it is not well retained after aldehyde fixation and the conditions of cell fixation should beconsidered carefully when this method will be applied.Nocodazole TreatmentNocodazole is an inhibitor of the cell cycle. It halts the cell cycle at the G2/M phase transition by disruptionof the mitotic spindles. When Nodocazole is added to a mammalian cell culture it results in the destructionof most cytoplasmic microtubules. The attachment of the spindle microtubules to the kinetochores ofchromosomes is essential in the development of the mitotic spindle, allowing for cell division. Since noneof these events can happen in the absence of microtubules the cell cycle will be halted at the end of G2.Nonyl Acridine Orange (NAO)NOA is a mitochondrial dye that can be used to investigate mitochondrial function. Its localization is notdependent upon the mitochondrial membrane potential. NOA has been used in the past to investigatedrug resistance and to measure the change in mitochondrial mass before/during apoptosis.Northern BlotA northern blot, from which the western blot technique is derived, has as main difference with the latterthat it detects RNA instead of proteins. mRNA is isolated and hybridized in northern blots. Before mRNA isloaded onto a gel to undergo electrophoresis it is first extracted and purified from the cell. mRNA is madeof different sizes and upon activation of an electric current through the gel, the mRNA will start to moveaway from the negatively charged electrode. This will separate the mRNA based on size after which alabeled probe for the mRNA sequence in interest will be introduced. After this the blot is washed and onlythe specific probe, usually labeled for detection, will remain bound.PerkinElmer Phos-tagThis method can be used to detect the phosphorylation of proteins. Phos-tag is a chelate that can mimicnatural phosphate binding proteins. It has great affinity for the phosphomonoesters of serine, tyrosine andthreonine. This tag can be used in a wide variety of detection methods.PerkinElmer Phos-tag StainsThis stain can be used to detect phosphoproteins in 2D polyacrylamide and SDS gels. It exhibits a highsensitivity with any imaging technique used. Staining can proceed at the natural PH of the protein withavoids the denaturing of proteins during the process.PerkinElmer Phos-tools KitKit combining PerkinElmer Phos-tag and Phos-trap methods.PerkinElmer Phos-trapIs used to measure phosphorylation of proteins. Phosphoproteins are cleaved in different peptides inpresence of titania-coated magnetic beads. The phosphorylated peptides bind to the beads, but theunphosphorylated ones do not. After this, further analysis with mass spectrometry can be used to identifywhich peptides were phosphorylated.Randox-Total Antioxidant Capacity KitUsed to standardize the total capacity of antioxidants within a solution. It is based upon the incubation ofABTS with metmyoglobin, which gives ABTS + . This ABTS + emits light at 600 nm. This emission, however,SCI 332 Advanced Molecular Cell Biology Research Proposal 99