experimental methods Flashcards
What are the two types of microscopy?
Light and electron
Light vs. electron
Light microscopy for larger subcellular components/organelles, electron microscopy for finer structures
What types of specimens can light microscopy be used on? Can it be labeled and if so with what?
Live or fixed specimen. Unlabeled or labeled with dye or fluorescence
Two types of electron microscopy and differences
TEM (transmission): 2D, visualizing structures within a specimen
SEM (scanning): 3D, visualizing 3d surface of specimen
What is GFP
A protein that contains a fluorophore
How does fluorescence microscopy work?
Sample stained with fluorescence, light shines on sample to excite the fluorophore
What is immunofluorescence
Conjugating antibodies with fluorophoes. Antibodies recognizes antigen (what we’re in interested in)
How do fluorophore tagged proteins expressed via transgenes work? Why would we use them?
Transfect cell with plasmid with GFP fused to our POI, makes it so that fluorescent fusion protein is produced upon expression.
How do geen expressional reporters work?
If one wants to study a certain sequence, such as a gene’s promoter, fuse a reporter gene that encodes GFP to the gene, which can reveal how gene expression is regulated (makes it easier to track)
What is differential centrifugation?
Separation of cellular material based on size and density
What do the different pellets of a centrifuged cell look like (from biggest to smallest)?
whole cells, nuclei, cytoskeletons –> mitochondria, lysosomes, peroxisomes –> microsomes, other small vesicles –> ribosomes, viruses, large macromolecules
What does SDS-page do?
Take centrifuged material, load them onto gel, sorts material by size on different lanes
What is the purpose of a pulse-chase experiment?
To track the activity of certain cells over a prolonged period of time
