L2 - Biochemical Techniques Flashcards
what should be the experimental approach of how something is being tested?
- compare cells w and w/o protein (or modified protein)
- experimental condition (stress)
how can cells be grown in culture?
1) adherent cells
2) suspended cells
adherent cells
cells grown on surface
suspended cells
cells grown in liquid
what is in cell culture media?
- minimal essential medium
- serum (growth factors so cell can live)
- antibiotics
how can you visualize cells?
microscopy
how can you manipulate proteins?
1) deletion (KO gene or -/-)
2) suppression (RNA interference with siRNA)
3) chemical inhibition (ex: phosphorylation manipulation)
ways to introduce exogenous material into cells?
1) microinjection
2) electroporation (open pores via short electric shock)
3) transfection (uncommon, but fuse vesicles containing substance X to PM to release material inside cell)
where does siRNA interfere?
siRNA binds to mRNA in cytosol and induces degradation
how is fluorescence microscopy used?
used to see spatial distribution of proteins in an adherent cell
what is an advantage of fluor microscopy?
can label multiple proteins in a cell at the same time and see potential overlaps
do you fluoresce at longer or shorter wavelength than the light absorbed?
at longer wavelengths (Stokes law)
what fluor tag to use to absorb UV light?
DAPI (fluoresces as purple-blue)
FITC
absorbs green and fluoresces green
rhodamine B
absorbs green and fluoresces orange
why can Abs be used as probes?
Y-shaped and can bind to specific proteins
describe immunofluorescence process
1) antigen bound to membrane
2) primary Ab binds to antigen
3) secondary Ab with marker (usually fluorophore) binds to primary Ab
why is detergent used in immunofluorescence?
allows Ab used as probes to enter cells
can cells be stained while adherent?
yes
can cells be stained while suspended?
yes
how is flow cytometry used?
quantify a large population of cells
describe flow cytometry
1) cells labelled with Abs in suspension
2) use laser to visualize the individual cells
3) get an output
what are the outputs of FACS?
1) histogram
2) dot plot
what does a histogram of FACS tell you?
the number of cells with a certain fluorescence intensity (represented by peak of curve) of each different population of cells in the sample
what does a dot plot of FACS tell you?
Side scatter (cell granularity) and forward scatter (cell size)
what is the main diff between fluor microscopy and flow cytometry?
fluor microscopy: stain adherent cells
flow cytometry: stain suspended cell populations
how is immunofluorescence used?
to find distribution of protein in tissue samples
