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Biomembrane Lab Techniques, e.g., PLs only, e.g., disruption via - Coggle…
Biomembrane Lab Techniques
How can we study membrane characteristics/membrane proteins in the
lab
?
Visualization
Electron microscopy :microscope:
Staining :lock_with_ink_pen:
Osmium tetroxide :speaking_head_in_silhouette:
Stains polar head
Annexin V :skull:
binding to phosphatidylserine
Generating
synthetic
bilayers
from
chemically
pure
phospholipids
:money_mouth_face:
lipid mixtures :green_salad:
preparation
Treat with
organic
(hydrophobic) solvent
selective
solubilization
of phospholipids and cholesterol :droplet:
Evaporate solvent
Disperse PLs in water
Spontaneously form liposomes
Dissolve PLs in solvent in container with partition :black_small_square:
planar bilayer over hole
proteins and carbohydrates :shallow_pan_of_food: remain in insoluble residue :white_small_square:
chloroform - methanol (3:1)
lipophilic dye: Congo red
Measuring movement
FRAP
:bulb:
phospholipids
fluorescent
substituent
diffusion coefficient
:runner:
rate at which membrane molecules move
proteins
antibody
tagged with fluorescent dye
Measuring relative abundance
PLs across leaflet
phospholipases
add to external medium
cannot cross the membrane
only cleave off PL head groups in leaflet it is next to
present in cytosol
PLC
Lipid rafts
Rafts enriched in glycolipids :honey_pot:
fluorescently labeled cholera toxin :microscope:
binds to certain gangliosides
methyl-β-cyclodextrin
extracts cholesterol
antibiotics (e.g.,
filipin
)
aggregates cholesterol
solubilize lipids and proteins
lipid-anchored
proteins
treatment of cells with
phospholipase C
cleaves phosphate-glycerol bond in PLs and in GPI & releases GPI-anchored proteins
Detergents
amphipathic
molecules
disrupt membranes by intercalating into PL bilayers
natural 🧂vs. synthetic (most)
ionic vs. non-ionic
ionic :warning:
contain a charged group
bind to
hydrophobic
regions of proteins
1 more item...
e.g., sodium deoxycholate & sodium dodecylsulfate (SDS)
at high concentrations, SDS completely
denatures
proteins (binding to every side chain)
1 more item...
non-ionic
e.g., Triton X-100 and octylglucoside
lack a charged group
bind to
hydrophobic
regions of proteins
2 more items...
at very low concentrations: detergents dissolve in pure water as isolated molecules
as concentration increases
can dissolve
membrane
proteins by coating
beyond
critical micelle concentration (CMC)
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charged head, lipophilic tail
peripheral
proteins
removal from PM
by solutions of high ionic strength (high salt concentrations), which disrupt ionic bonds
by chemicals that bind divalent cations like Mg2+
most are soluble in aqueous solution
don't need to be solubilized by detergents
e.g.
PLs only
e.g.
disruption via