supplementary pracitcal Flashcards
chemical hazard
bacterial lysate
acrylamine in the gel is a neurotoxin and carcinogen
SDS- PAGE separates proteins by
molecular weight
what does SDS-PAGE stand for
sodium dodecyl sulphate - polyacrylamide gel electrophoresis
insertion of a novel gene into different expression vectors can increase
expression of the protein
how much of our samples do we need to add to SDS gel
can work out from BSA standard curve
4 E.coli bacterial strains
stain A
A - expression plasmid with insert, expression regulated by IPTG
periplasm
space between two membranes
strain B
B - as for A but with a periplasmic tag (tag attached to N-terminal and translocated protein to periplasm)
strain C
expression under bacterial control (no promoter in plasmid)
IPTG
switches on the promoter region, increasing expression of protein
stain D
no expressed protein
6 samples to analyse
AC - strain A cytoplasm
AP - strain A periplasm
BC - strain B cytoplasm
BP - strain B periplasm
CT - total protein strain C
DT - total protein strain D
how to get the samples from the bacteria
centrifugation
sonication - sound waves breaks and disrupts the bacteria
why periplasm
not many proteins in this region so if we traffic our protein there itll be more easier to purify our protein and easy to identify it
BSA
BSA is the universally accepted reference protein for total protein quantitation
graph shows the optical density of known BSA concentrations
compare optical density of our proteins using bradford reagent to work out protein concentration
10x dilution factor
so concentration needs to be multiplied by 10 to get results
SDS-PAGE
separation of proteins in an acrylamide gel by molecular weight
proteins denatured by the presence of SDS that gives the protein a negative charge therefore move upward towards the anode (red)
DTT breaks
disulphide bonds
Rf =
distance moved by protein / distance moved by dye front
plot Rf of y axis
plot log10 MW on X axis
the beer lambert law
A = Ecl
A = absorbance
E = extinction coefficient
C = concentration
L = path length (cm)
want 20 microliters so do 20/protien conc from BSA graph
5.24mg/ml = 5.24 ug/ul
need 20ug so 20/5.25 = 3.819ul (4 ul)
T7 promoter is controlled by
IPTG
novel gene is in what expression vector in strain A
pET21a
novel gene is in what non- expressing vector in stain C
pUC18 (non expressing wont make our protein)
pET21a
ampicillin resistance
lacl - lac repressor
has own promoter
multiple cloning site (Multiple cloning sites are a feature that allows for the insertion of foreign DNA without disrupting the rest of the plasmid)
IPTG binds to lac repressor and pulls the repressor off the lac operator allowing it to work allowing full expression T7 and protein
