Working With Nucleic Acids + Proteins Flashcards
what do you need to think about when working with cells + tissues?
- temp effects
- microbial contamination
- changes in sample content (metabolic reactions/breakdown)
- is container/bag changing the sample
describe cell isolation?
- remove culture supernatant + add pre-warmed trypsin EDTA to cell monolayer
- add equal vol of complete medium
- to neutralise trypsin + collect cells in tube
- centrifuge at 200 x for 3 mins to pellet cells
- remover supernatant
- plate cells once resuspended with culture medium into culture vessels
what is trypsin?
enzyme that breaks down proteins
what is EDTA?
chemical that chelates/removes divalent cations
describe cell transformation/engineering?
- DNA/RNA + transfection reagent
- incubate for 20 mins
- transfection complex added to cells
- assay cells 24-72 hours post transfection
describe differential centrifugation?
- heterogenous sample added to vessel
- sample separated via centrifugal force
- sample separated into pellet and supernatant sample
what can differential centrifugation be used to separate?
- cells from cell growth media
- precipitates from soluble fractions
describe rate-zonal centrifugation?
- heterogenous sample added to vessel with sedimentation gradient matrix
- sample separated via centrifugal force - into discrete zones of tube/matrix
- sample separated into sediment fractions + particles isolated from each fraction
what can rate-zonal centrifugation be used to isolate?
- cell structures/organelles
- proteins/rna/dna based on shape/size
cell lysis techniques
- purify proteins to disrupt tissues + cells
- homogenise plasma membrane to rupture cells
- homogenate has large and small mol from cytosol e.g. enzymes, ribosomes etc
what are the 4 common procedures used to rupture the plasma membrane of cells?
- break cells with high frequency sound
- use mild detergent to make holes in membrane
- force cells through small hole using high pressure
- shear cells between close fitting rotating plunger + thick walls of glass vessel
what are the physiochemical properties of proteins?
- contain C, H, O, N
- have defined structure/shape
- have defined mass
- absorb light in UV range
- have both charged + uncharge AA
- have hydrophilic + hydrophobic AA
proteins in a solution absorb UV light with an absorbance maximum of 280nm
what is the reason for this?
due to the aromatic AA
phenylalanine, tryptophan, tyrosine, histidine
what is A280 (UV absorption) used for?
detect proteins during purification
routinely used to detect proteins on chromatographic separation
what law can you use to determine protein concentration?
beer Lambert law
describe the action of large proteins in size exclusion chromatography?
pass through column first
as have shorter flow path through column
in Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis, what is the gel?
porous mesh of acrylamide fibres
what are proteins given in Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis?
overall net neg charge by coating them in SDS detergent
describe the action of proteins in Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis/
- large proteins find it difficult to move through mesh —> take longer
- smaller proteins move more quickly
- reducing agents added to sample to break S-S bonds - unlink different PPC
the electrophoretic mobility of a protein on SDS polyacrylamide gel is related to its…
Mr
western blotting
- used detect specific proteins after SDS PAGE separation
- using antibodies to proteins of interest
- proteins transferred to membrane
- membrane probed for specific proteins with antibodies (usually carrying chemiluminescent tag)
what are the different types of chromatography?
- gel filtration
- antibody-affinity
- ion-exchange
for the Edman degradation, after how many AA can you usually identify the protein?
10
where does the stability of nucleic acids come from?
stacking interactions + water exclusion from internal bases
