Tour-de-Force

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Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007transport chain. Although they both inhibit mitochondrial activity, these inhibitors have different effects onmitochondria and the cell.Ion Trap Tandem Mass Spectrometry analysisTandem mass spectrometry is a more advanced version of the single mass spectrometry. In ion traptandem mass spectrometry, ions are trapped at the same place and several separation steps occur overtime. Several spectrometry measurements can be performed on the mixture, whereas in original versiononly one type of measurement would be possible.JC-1 Dual-Emission Potential-Sensitive ProbeThis is a potential sensitive probe with a dual-emission that can be used to measure mitochondrialmembrane potential. The probe is green when the mitochondrial membrane potential is low and it turnsred at higher membrane potentials. The ratio of the two colors is a measure of mitochondrial membranepotential. This probe specifically measures membrane potential and is not affected by other mitochondrialfactors.Kinase AssayProtein kinases phosphorylate other proteins by catalyzing the addition of a phosphate group to a certainlocation of the proteins. As regulation by phosphorylation is very significant in different functions of the cell,measuring activity of these kinases can prove to be important. In this technique, a certain kinase protein isintroduced to the medium together with ATP and the substrate that the kinase is known to act upon.Phosphate groups are tagged with radioactive or fluorescent tags and become active once incorporatedinto the substrate. Measurements of the activity of these tags indicate protein kinase activity.Knockout/Inhibition of Cyclins/CDKsA knockout of a protein is used to inhibit its activity. The cell is transfected by a plasmid or a DNAconstruct. This transfected construct recombines with the gene of interest. A sequence from the gene istransferred into the construct and thus a part of the original gene is deleted. The deletion of the sequenceprevents proteins from being translated. Even if the proteins are translated, they are non-functional. Thistechnique is used widely to study proteins whose functions are unidentified.Mass SpectrometryMass spectrometry can be used to identify compounds in a given mixture to determine the structure of thecompound as well as to measure the amount of compound in that mixture. The basis of this technique ismeasuring the mass to charge ratio of ions.MitoSOX Red ProbeROS is inevitable as a side product of cellular respiration in aerobic organisms. O 2-superoxide ispredominant in mitochondria and its presence initiates a cascades producing more ROS, includinghydroxyl peroxide and hydroxyl radical.To monitor O 2-in living cells, hydroethidine (HE), which is a reduced form of the nucleic acidintercalator ethidium, is used as a indicator of ROS. Once HE is oxidized, it exhibits weak cytosolic bluefluorescence. However, the probe also binds to nucleic acids, which results in staining the nuclei andparticularly the nucleoli by red fluorescence. The MitoSOX Red mitochondrial superoxide indicatorconsists of HE covalently bound to a triphosphonium cation via a hexyl carbon chain. The phoshoniumgroup is positively charged and targets HE analog to mitochondria. Here it accumulates as a function ofmitochondrial membrane potential after which it exhibits red fluorescence due to oxidation and binding tonucleic acids in the mitochondria.So far, ethidium has been postulated as the oxidation product of HE. Recent findings, however,indicate that 2-hydroxyethidium is the oxidation product of HE. The conventional wavelength that is usedto indicate ethidium was 510 nm. By using this wavelength is was hard to distinguish between ethidiumand 2-hydroxyethidium and therefore it is more significant to use a wavelength of 396 nm to excite theMitoSOX red probe. The MitoSOX Red probe with 396 nm excitation can be used to selectivelymonitor O 2 - in the mitochondria.SCI 332 Advanced Molecular Cell Biology Research Proposal 98
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
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