interview Flashcards
(33 cards)
When do we harvest cells?
When cells reach desired density. They are then moved to the downstream process.
Downstream process outline (3)
1) cell capture and disruption
2) intermediate purification
3) polishing
4) mixed with inert ingredients, packed, sent to market
Cell capture and disruption
quick separation of product from cells in the bioreactor: removes majority of water, enzyme, etc
Cell capture and disruption - intracellular
keep pellet,
disrupt cell to release product through: centrifugation, filtration
Cell capture and disruption - extracellular
keep media, discard pellet (ultrafiltration, centrifugation)
Intermediate purification
removal of bulk contaminants (viruses, endotoxins, etc)
Polishing
elimination of trace contaminants and impurities. Ex: partially unfolded peptide chain. Purity from 98% to 100%
Purification Techniques (7)
1) centrifugation,
2) SDS-PAGE,
3) BCA assay,
4) Spectrophotometer,
5) Western Blot,
6) Filtration (TFF)
7) Chromatography
SDS-PAGE purpose
to isolate proteins by size
How are proteins converted to the same shape? (3)
1) SDS detergent,
2) beta-mecarptoethanol
3) heating samples to 95 degrees celsius for 5 min
(SDS) detergent
solubilizes proteins to linear shape and gives them many negative charges. It disrupts secondary, tertiary structures.
(SDS) beta-mecarptoethanol
reduces disulfide bonds
(SDS) why is a sample of protein boiled in SDS and beta-mecarptoethanol?
Heat shakes up molecules allowing SDS to bind to hydrophobic regions and complete denaturation.
SDS-PAGE loading buffer ingredients (5)
1) Tris-HCl: gives appropriate pH and helps samples move faster through gel,
2) SDS detergent: denatures proteins
3) Glycerol: makes samples viscous and sink into wells,
4) Bromphenol Blue: tracking dye, helps visualization of bands on gel,
5) Coomassie Brilliant Blue: stains gel after it’s done
What type of information can you get from SDS-PAGE?
1) size,
2) relative concentration,
3) protein standards give you an estimate
SDS-PAGE outline (11)
1) prepare gel,
2) seal gel in place with agarose and check for leaks,
3) mix your protein samples and loading buffer,
4) heat all samples to 95 degrees celsius for 5 min,
5) load samples into wells,
6) turn on apparatus at 150 volts until bands move about halfway, then 200 volts until the end,
7) turn off apparatus,
8) stain gel with Coomassie Brilliant Blue,
9) place gel on shaker,
10) destain gel,
11) photograph gel.
Western Blotting use (4)
1) after SDS-PAGE,
2) gives MW,
3) it’s specific to the target protein,
4) detects very small amount of protein.
Western Blot outline (6)
1) separate proteins by SDS-PAGE,
2) semi-dry apparatus: transfer proteins from gel to nitrocellulose membrane (mirror image: effective transfer)
3) block nonspecific sites on membrane with blocking buffer (5% dry milk powder) so only the target protein will stick to membrane,
4) incubate with primary antibody, wash unbound antibodies,
5) incubate with secondary antibody, wash unbound antibodies,
6) enzyme attached to secondary antibody converts: soluble, colorless substrate to colored, insoluble product. colored bands appear where protein interacts with primary antibody,
What is the conjugated enzyme used in western blot?
(AP) alkaline phosphatase or horseradish peroxidase
Calcium competent cells
to increase prokaryotic cells ability to incorporate plasmid DNA so it can be transformed later. (heat shock)
Bioreactor (definition)
an apparatus to grow organisms under controlled conditions. used to produce pharmaceuticals, vaccines, antibodies, etc
Parameters to monitor bioreactor: (4)
1) pH,
2) temperature,
3) agitation,
4) oxygen concentration
Parameters to monitor cell growth (
1) microscopic count,
2) growth curve,
3) optical density
4) spread plates,
5) gram stains,
6) fixed volumes were taken at certain intervals
Pichia pastoris (3)
1) high density biomass,
2) secretes recombinant protein into media,
3) doesn’t secrete many endotoxins (easier purification)
