Cell Bio 2 Flashcards
(14 cards)
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 (Hoechst stain)
Cells with bound antibodies or that have taken up the dyes can now be sorted and counted.
FACS Machine
Fluorescent
Activated
Cell Sorting
FACS Machine steps
- cells pass single file through a laser light beam
- Both fluorescent light emitted and scattered are measured by detectors
- Individual cells are forced thrugh a nozzle and given a change proportional to the degree of fluorescence detected
- Cells with different electric charges are seperated by an electric field and collected.
Quantification of cells expressing two different cell surface markers by FACS
As the cells pass through the FACS machine the intensity of green and red fluroresence emitted by each cell is recorded.
Cells Cycle Analysis by FACS
Cells that have replicated their DNA but not fully divided (G2) will have twice the Hoechst stain fluorescence intensity of non-dividing cells (G1)
How to Isolate Cell Organelles (Step 1)
Mechanical homogenization Sonication (ultrasound) Pressure (cells are forced through a very narrow valve) Non-ionic detergents placing cells in hypotonic solution
Step 2
Centrifugation of Cell Homogenate:
i) differential
ii) equilibrium density-gradient
Differential Centrifugation
Spinning homogenate yields pellet and supernatant.
Increasing centrifugal force gravity to isolate organelles.
Nucleus pellets first Mito + chloro + lysosome plasma membrame + microsome Ribosomal subunits soluble part od the cytoplasm
the heavier the organelle the weaker the g force necessary to pellet the organelle.
Equilibrium Density-Gradient Centrifugation
Seperation based on density
Homogenate is applied to a gradient sucrose
At high speed/several hours organlles migrate to sucrose layer equal to their own density and remain there
How are proteins seperated from the organelle?
Non-ionic detergents: disrupts lipid bilayer
Ionic Detergents: denaturing and Disrupts ionic and H-bonds
Detergents have an amphipathic nature a hydrophobic properties and hydrophilic properties, the hydrophobic domains can penetrate into the lipid bilayer, chemicals can integrate into the phospholipid bilayer and disrupt the membrane.
SDS
Highly charged sulfate head and ionic charge (SDS), SDS is used to denature proteins and unfold them.
SDS is denaturing and binds to and destabilizes the hydrophobic side chains within the core of proteins
SDS-PAGE
Electrophoretic separation of proteins.
Gel wedged between two glass plates apply an electric field.
PAGE is carried out in the presence of negatively charged detergent SDS.
Polypeptide chains are forced into extended negatively charged conformations with a similar charge-to-mass ration.
The mobility of SDS-portien complexes are influenced primarily by molecular size
Speed of travel
Is inversely proportional to the size of the protein
Western Immunoblotting
Gel is loaded onto the sandwich cassette (immunoblotting apparatus)
the blot is placed on top and then placed into the electroblot apparatus.
The proteins are transferred onto the blot membrane from the gel (after an electric current is applied).
Proteins can be probed with antibodies.
To detect the primary antibodies we add a secondary antibody that bonds to the primary antibody the most common type of secondary antibody is conjugated to an enzyme
We add substrate (luminol) horse radish peroxidase will catalyze a reaction with luminol, it causes a local production of luminescence where the antibodies are found.
Bands start to glow.
Modern approaches prefer fluorescent-labeled antibodies with special illuminating cabinets that cause fluorochrome bound tot antibodies to fluoresce.
