Tour-de-Force

Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007Cell FractionationCell fractionation refers to a broad set of techniques aiming to separate and isolate specific organellesfrom cell. In other words, it is the separation of homogenous sets (organelles) from a heterogenouspopulation of cells. There are a number of steps involved in cell fractionation; namely disruption (alsoknown as homogenization) of cells, macro-filtration and purification of the cell components.Central-ElutriationCentrifugal elutriation is a technique used to synchronize cells at specific phases of the cell cycle. It is adrug-independent method capable of synchronizing cells from asynchronous cell populations in any phaseof the cell cycle. This method entails that cells in a particular phase in the cell cycle can be isolatedaccording to their size. In other words, early G1 cells are around half the size of cells in late G2 or Mphase. However, it is important to consider that not all cell types show significantly enough size variabilitythroughout the cell cycle.Cerium Ions with ReflectanceConfocal laser scanning microscopy adaptations have been made to allow detection and assessment ofsemiquantitatively cerium based primary reaction products of oxidases, phosphatases as well as the 3,3-diaminobenzidine (DAB) based primary reaction product of cytochrome-c-oxidase. This technique offers aunique method to make histochemical reaction products, which are weakly absorbent, but sufficientlyreflective, visible.ChemiluminescenceChemiluminescence is the generation of light by a chemical reaction. When used as a method, a specificprobe can be used, which reacts with a specific compound. The luminescence can then be measured by aluminometer, to show and quantify the presence of the compoundCo-immunoprecipationCo-immunoprecipation can be used to check if proteins physically interact with each other and form animmune complex. First, the cell gets lysed, after which antibodies get added, which specifically bind to theprotein complex. Then the antibodies and the bound immune complex get immobilized on a solid complex,after which the remaining cell lysate is washed off. The complexes are then eluded and analyzed by SDS-PAGE usually followed by a western blot.Figure A.1: Principles of co-immunoprecipitationSCI 332 Advanced Molecular Cell Biology Research Proposal 96