Week 3 Flashcards
P purification analysis
physical and chemical properties :
Mass/size (&shape)
Density
Electrial charge
Binding affinity
separation methods:
Centrifugation
Electrophoresis
Chromatography
Centrifugation
Rapid turning of tube= centrifugal F measured by earth’s gravity(=1g)
Force acts on particles suspended within the liquid medium of the tube
rate of supernatant clearance depends on
size/mass…Calculated by the Svedburg (s)
centrifugation density and types
Parties denser (usually) = g force will push them to bottom
Particles less dense = g force will cause floating toward the top
if same density as medium= no mvt
swinging bucket rotor
fixed-anglw rotor
differential centrifugation
seperation of contents by size/mass
low speed centrifugation pellets nuclei
leaves mitho in the supernatant
can be recovered as pellet by higher-speed cent.
purification of coronavirus
Infected cell homogenate
First spin: Medium-speed= pellet nuclei, mithoch
Second spin: High-speed=pellet virus particles
Resuspended pellet, apply equilibrium density gradient
Equilibrium density gradient centrifugation
high -) low density gradient of medium
Put pellet on top
centrifuge at high speed
Virus starts to sink but stops when solution p= virus p
electrophoresis (masse/size ratio)
Direction of migration determined by net charge
Speed of migration determined by net charge/mass ratio
Faster if mass is small(small=less interactions=faster)& charge high
migration rate in inv. prop to P size
SDS
The anionic detergent sodium doucecyl sulfate (SDS)
hydrophobic and hydrophilic endx… hydrophobic gets in P to bind P hydrophobic part=disruption of oil drop… SDS bind to itself too and due to negative charge repulsion… it causex the unfolding of P
post transl. mol.
Post-transl. mod. might have
impact on mobility during sDs
polycrylamide gel electrophoresis
Mobility effect result from phosphate gr.
locally interfering us sps binding
Phosphorylation = fewer sDS=
fewer charge=slower
isoeletric focusing
isoelectric point (PI): pH at which sum of charges is 0
depends on a.a. comp. of each P
Ph gradient immobilized in oxidized in acrylamide gel
P in an electrical field migrate and stops at their own PI
pH
low ph… acid=neutral/ basic=+
high pH… acid=-/basic=neutral
No relationship between PI & molecular weight,
so separated P spots are widely
distributed= many diff P
mass spectrometry
Analytical (not preparative) method(P is destroyed)
High-precision determination of the charge/mass ratio of ionized mol
concept (3)
concept of mass spectrometry
1) Produce dispersed (indiv.mol) ions in a gas phase because P can’t “fly”
2) Measure the a of ions in electric magnetic field
3) Acc.depends on mass/charge ratio (m/z)
if mol.= single charge… m/z=mw (mol. weight)
electrospray ionization
the gas-phase ions generated by electrospray are separated in mass analyzer into sep. pop. differing in m/z
MS/ MS (Tandem MS)
Recovering an ion, fragmenting it by high E collision w/ an inert gas & doing mass spec. on fragments
pick a peptide ions from MS & tune the field so it doesn’t read but zip it & smash then into fragments partially & randomely
Gives info about sequencing while comparing mol weight
“second dimension”of info
(analyzed computationally)
can identify the a.a. sequence
of the peptide ions
Proteomics=analysis of biol. P
by mass spec.& bioinformatics
foidentify the pop. of P present
in any subcellular organelle
(unique solution)
chromatography
Separation of components based on differential intractions w/ immobile solid material
Mobile phase liquid or gas…generally aqueous buffer
move continously past solid phase
P mol. mvt rate depend on how much they interact w/ solid phase
Diff chromatography methods based on diff. kinds of
interactions of P w/ solid phase; generally done in columns
