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Lecture 3

Lecture 3 - Isolation and Analysis of Cell Organelles and Molecules

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Biology 2382B
Sashko Damjanovski

LECTURE 3: ISOLATIONANDANALYSIS OF CELL ORGANELLES AND MOLECULES Labeling live cells with fluorescent antibodies or stains • Antibodies made against specific cell surface proteins can be linked to fluorophores • Membrane permeable fluorescent dyes can be used to label intracellular structures (i.e. Hoechst stain binds DNAin nucleus) • Cells with bound antibodies or that have taken up the dyes can now be sorted and counted • Antibodies are used to isolate pure fractions of cell • Note: fluorophore-linked secondary antibodies could also be used in combination with unlabeled primary antibodies FluorescentActivated Cell Sorting (FACS) 1. Cells pass single file through a laser light beam 2. Both fluorescent light emitted and scattered are measured by detectors 3. Individual cells are forced through a nozzle and given a charge proportional to the degree of fluorescence detected 4. Cells with different electric charges are separated by an electric field and collected • FACS can both analyze the cells and select one or few cells from thousands of others and sort them into a separate culture dish. To sort the cells, their concentration has to be adjusted so that the tiny droplets that the FACs machine makes and analyzes contain only one cell each.Astream of droplets is analyzed for fluorescence and those that have the desired signal are sorted away from those that do not. The cells will only fluoresce if excited. Having been sorted from other cells, the selected cells can be grown in culture. • FACS separates cells that are labeled differentially with a fluorescent reagent o Step 1: a concentrated suspension of labeled cells is mixed with a buffer (the sheath fluid) so that the cells pass single-file through a laser light beam. o Step 2: both the fluorescent light emitted and the light scattered by each cell are measured; from measurements of the scattered light, the size and shape of the cell can be determined o Step 3: suspension is then forced through a nozzle, which forms tiny droplets containing at most a single cell; at the time of formation at the nozzle tip, each droplet containing a cell is given a negative electric charge proportional to the fluorescence of that cell determined from the earlier measurement o Step 4: droplets now pass through an electric field, so that those with no charge are discarded, whereas those with different electric charges are separated and collected Quantification of cells expressing two different cell surface markers by FACS • As the cells pass through the FACS machine, the intensity of the green and red fluorescence emitted by each cell is recorded • Each dot represents a single cell • The proportion of each cell population can be calculated Cell CycleAnalysis by FACS • Cells that have replicated their DNAbut not fully divided (G2) will have twice the Hoechst stain fluorescence intensity of non-dividing cells (G1) • Cells that have a diploid number will have a certain Hoechst stain intensity. Cells that start from replicating increase intensity and number of cells. We can quantify proportion of cells at different phases of the cell cycle. • G1 is the longest phase of the cell cycle, and so most cells are in this phase. How to study organelles and their proteins? • All eukaryotic cells contain membrane-limited compartments called organelles • The isolation of organelles is based on their different density and sizes • Among cell organelles, the nucleus, mitochondria, and chloroplast are bounded by two bilayer membranes • All other organelles are surrounded by single membrane How to isolate cell organelles? • Step 1: disruption of cell plasma membrane o Mechanical homogenization – plunger is forced into tube, cells are squished and forces cells to break apart through such a narrow channel o Sonication (ultrasound) – using high frequency such as ultrasound, you can break apart cell membranes o Pressure – cells are forced through a very narrow valve o Non-ionic detergents (i.e., Triton X-100) – intercalates the phospholipid bilayer and pokes holes in it, outer membrane becomes leaky o Placing cells in hypotonic solution – water will rush into cell which causes the cell to swell up and eventually the plasma membrane ruptures • Step 2: centrifugation of cell homogenate o Differential o Equilibrium density-gradient New and Used Centrifuges • Centrifugation allows for subcellular fragmentation. The spinning rotor interacts with air molecules and heats up.Air needs to be evacuated out and the rotor also needs to be cooled. • Centrifuges need to be balanced, so that equal weight is carried all around on the rotor o No balancing = wobbling (this is bad) • Safe centrifugation requires balanced loading of the centrifuge rotor. Imbalanced rotors can lead to damage to centrifuge and rotors Differential Centrifugation • Spinning homogenate yields pellet & supernatant • Increasing centrifugal force (gravity) to isolate organelles based on mass • Most cell-fractionation procedures begin with differential centrifugation of a filtered cell homogenate at increasingly higher speeds. • After centrifugation at each speed for appropriate time, the liquid that remains at the top of the vessel, the supernatant, is poured off and centrifuged at a higher speed. • The pelleted fractions obtained by differential centrifugation generally contain a mixture of organelles, although nuclei and viral particles can sometimes be purified completely by this procedure • Differential centrifugation is a common first step in fractioning of a cell homogenate. Differential centrifugations means centrifuging a homogenate at different speeds and times. Cell organelles which differ in size and mass travel to the bottom of the centrifuge tube at different rates • The homogenate resulting from disrupting cells is usually filtered
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