L2 - Biochemical Techniques

Question 1

what should be the experimental approach of how something is being tested?

Answer 1

• compare cells w and w/o protein (or modified protein)

- experimental condition (stress)

Question 2

how can cells be grown in culture?

Answer 2

1) adherent cells

2) suspended cells

Question 3

adherent cells

Answer 3

cells grown on surface

Question 4

suspended cells

Answer 4

cells grown in liquid

Question 5

what is in cell culture media?

Answer 5

• minimal essential medium
• serum (growth factors so cell can live)
• antibiotics

Question 6

how can you visualize cells?

Answer 6

microscopy

Question 7

how can you manipulate proteins?

Answer 7

1) deletion (KO gene or -/-)
2) suppression (RNA interference with siRNA)
3) chemical inhibition (ex: phosphorylation manipulation)

Question 8

ways to introduce exogenous material into cells?

Answer 8

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)

Question 9

where does siRNA interfere?

Answer 9

siRNA binds to mRNA in cytosol and induces degradation

Question 10

how is fluorescence microscopy used?

Answer 10

used to see spatial distribution of proteins in an adherent cell

Question 11

what is an advantage of fluor microscopy?

Answer 11

can label multiple proteins in a cell at the same time and see potential overlaps

Question 12

do you fluoresce at longer or shorter wavelength than the light absorbed?

Answer 12

at longer wavelengths (Stokes law)

Question 13

what fluor tag to use to absorb UV light?

Answer 13

DAPI (fluoresces as purple-blue)

Question 14

FITC

Answer 14

absorbs green and fluoresces green

Question 15

rhodamine B

Answer 15

absorbs green and fluoresces orange

Question 16

why can Abs be used as probes?

Answer 16

Y-shaped and can bind to specific proteins

Question 17

describe immunofluorescence process

Answer 17

1) antigen bound to membrane
2) primary Ab binds to antigen
3) secondary Ab with marker (usually fluorophore) binds to primary Ab

Question 18

why is detergent used in immunofluorescence?

Answer 18

allows Ab used as probes to enter cells

Question 19

can cells be stained while adherent?

Answer 19

yes

Question 20

can cells be stained while suspended?

Answer 20

yes

Question 21

how is flow cytometry used?

Answer 21

quantify a large population of cells

Question 22

describe flow cytometry

Answer 22

1) cells labelled with Abs in suspension
2) use laser to visualize the individual cells
3) get an output

Question 23

what are the outputs of FACS?

Answer 23

1) histogram

2) dot plot

Question 24

what does a histogram of FACS tell you?

Answer 24

the number of cells with a certain fluorescence intensity (represented by peak of curve) of each different population of cells in the sample

Question 25

what does a dot plot of FACS tell you?

Answer 25

Side scatter (cell granularity) and forward scatter (cell size)

Question 26

what is the main diff between fluor microscopy and flow cytometry?

Answer 26

fluor microscopy: stain adherent cells

flow cytometry: stain suspended cell populations

Question 27

how is immunofluorescence used?

Answer 27

to find distribution of protein in tissue samples