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Biology 2B03 > Week 4 + Module 3 > Flashcards

Week 4 + Module 3 Flashcards

(51 cards)

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1
Q

Why extract and purify proteins

A
  • structural/functional analysis
  • determination of amino sequence
  • development of antibodies to a protein
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2
Q

Steps to purifying proteins

A
  1. Get good assay
  2. Select protein source
  3. Break open cells - extract protein
  4. Solubilize protein
  5. Stabilize protein
  6. Fractionate - separate
  7. Determine purity
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3
Q

Protein assay

A
  • should be specific to the protein’s unique characteristics

ex.
- enzyme activity (use of substance)
- antibody (if protein is there)
- biological activity/function (if it binds)

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4
Q

Sourcing and extracting protein

A

Get it from somewhere where it
- is easily obtainable in large amounts
- contains a high concentration of the protein of interest
- low in proteins that may co-purify
- low in proteases

Extract through
- breaking/lysing cells
- chemical lysis
- physical grinding
- ultrasonic sonicators to break biomembrane

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5
Q

Solubilize protein

examples of solu/insolu

A

Soluble
- cytosolic and secreted proteins
- polar
- detergents present

Insoluble
- transmembrane proteins
- membrane-associated pros
- amphipathic/non-polar

pH or salt content also have an affect

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6
Q

Stabilize protein (maintain native structure)

A

pay attention to
- not hot temperature
- protease inhibitors
- ligands
- salts
- metal ions
- not high concentration
- pH

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7
Q

Protein fractionation

based on

A
  • proteins can be separated based on size, polarity, charge, solubility, shape
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8
Q

Protein fractionation - techniques

A

Charge
- ion exchange chromatography
- gel electrophoresis

Size
- gel electrophoresis
- gel filtration chromatography
- ultracentrifugation

Polarity
- absorption chromatography
- hydrophobic interaction chromatography

Specificity of binding
- affinity chromatography

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9
Q

Separation step 1 - differential centrifugation

A
  • separate based on size or location within a cell
  • keep increasing g = centrifugal force
  • things accumulate in pellets at the bottom
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10
Q

Separation step 2 - separation by chromatography

A
  • column chromatography used
  • more interaction btwn protein and beads = moves slow
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11
Q

Ion exchange chromatography

A

in this case OPPOSITES attract

  • to remove, wash with interacting thing (warm, salt, pH)
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12
Q

Gel filtration chromatography

(orbeez)

A
  • small molecules fit in indents of beads
    = travel slower
  • remove by washing or spinning

threshold size = largest protein that could fit

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13
Q

Affinity chromatography

A
  • proteins recognized by antibody bind to them (affinity)

= don’t elute until you disturb interactions (pH or heat)

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14
Q

SDS-PAGE electrophoresis

A
  • denature proteins with sodium dodecylsulfate (eliminates shape)
  • add (-) detergent that associates with all proteins (eliminates charge density effect)
  • purify (centrifuge, ion, gel filter, affinity)
  • electrophoresis using western blot
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15
Q

Western blot

A
  • blot/transfer to a membrane
  • use specific antibodies to identify protein of interest
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16
Q

Activity vs. specific activity

A

activity
- all proteins

specific activity
- just protein of interest

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17
Q

Light microscopy

A
  • uses visible light
  • conventional and fluorescent
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18
Q

Electron microscopy

A
  • transmission and scanning
  • beam of electrons
    objects less than 200 nm
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19
Q

Resolution or limit of resolution (D)

A

D = 0.61λ / NA

D = minimum distance between two objects that can be distinguished from one another

λ = wavelength
shorter = higher res. (smaller D)

NA
= numerical aperture (want this to be =1)
- lets us gather light and see detail

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20
Q

Brightfield microscopy

A
  • live (from transparent things)
    OR fixed (dead things)
  • stained OR unstained
  • uses light
  • is colourful (purple-y)
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21
Q

Nomarski and phase contrast

Study These Flashcards
A

Nomarski (like a fossil, “no mar”)
- 3D imprint
- edges and surface of cell structures

Phase contrast
- halo around them
- internal cell structures

Both unstained and unfixed (live) specimens if transparent
- both enhance density/refractive difference

22
Q

Immunofluorescence microscopy

Study These Flashcards
A
  • fixed/dead samples
  • molecules of interest stained with fluorescent dyes or tagged with fluorescent antibodies
  • locating specific cell molecules

detect+localize

23
Q

Tagging proteins with GFP

Study These Flashcards
A
  • live / dynamic samples

GFP = green fluorescent protein

  • recombinant fluorescence gene
24
Q

Confocal scanning microscopy

Study These Flashcards
A
  • high-resolution images from fluorescently labelled samples
  • excites only fluorophores in a specific section
  • eliminate background/surrounding fluorescence
25
Deconvolution microscopy
- subtracts blurry layers using computer software
26
TEM
- beam of electrons at thin section - look at internal structures in detail 0.1 nm resolution limit
27
SEM
- beam of electrons at solid specimens - reveals info about surface and texture in 3D
28
Biomembrane facts
- define compartments with unique internal environments - hold signaling/adhesion proteins - are flexible and dynamic - receptors to sense enviro - selectively permeable - grow and change cell shape - anchor microtubules Organelles - luminal and cytosolic faces Cell - cytosolic and endoplasmic faces
29
Biomembrane constitution
outer leaflet mostly: - sphingolipids - cholesterol-rich microdomains inner leaflet - cholesterol and GPLs
30
Phospholipid bilayer / micelle
- hydrophilic head - hydrophobic tails = are amphipathic - assemble spontaneously micelle; like a flower of matchsticks with tips out
31
Phospholipid
Hydrophile phosphate head 2 hydrophobic fatty acid tails - diglycerides
32
Fluid mosaic model
- lateral movement through the membrane = fluid - mad of many different macromolecules = mosaic
33
Leaflets of the bilayer
x2, one facing in one facing out - have distinct characteristics each one layer of phospholipids ooooooooooooo |||||||||||||| |||||||||||||| ooooooooooooo
34
FRAP measuring fluidity
Fluorescence Recovery After Photobleaching - membrane proteins fluorescently labelled with fluorophores - laser saturates some so they no longer fluoresce - diffusion of fluorescent proteins back into photobleached area
35
FRAP - calculating fluidity
- look at drop when it's bleached - see intensity it returns to 100% back to same intensity as original 50% regains half the fluorescence of the bleach drop
36
What makes membrane more or less fluid
Less fluidity - Saturated acyl fats - Long acyl chains (get tangled) - Colder More fluidity - Unsaturated (C=C) kinky - Short acyl chains - Hotter
37
Regulating membrane fluidity depending on temperature
Too hot - add cholesterol Too cold - add cholesterol - cleave fatty acids C18 to C16 - activate desaturase enzyme to make more unsaturated C=C Cholesterol - separates fatty acids so they don't crystalize in cold temps - decreases fluidity in hot temps
38
Cholesterol - phase separation
prefers sphingolipids to glycerophospholipids - favours phase separation where sphingos are away from gpls v^v^v^ ^ = cholesterol v = sphingo (cone shaped) GPLs (cylindrical)
39
Lipid rafts
microdomains - longer fatty acid chains - more cholesterol = less fluid = molecules are less mobile in there
40
Lipid rafts - function
biology - signal transduction - changes in shape during migration - adhesion rafts - immune cells - form blood-brain barrier disease - cancer cells have less asymmetry of lipids and more lipid rafts - amyloid plaque accumulation - Alzheimers - rafts can be targets different pathogens - prions can use to spread
41
Function of lipid rafts - cancer
Normal - more phosphatidylcholine (PC) and sphingomyelin (SM) in outer leaf - more phos-serine (PS) and phos-ethan-amine (PE) in inner leaf Cancer - more evenly distributed = less asymmetrical
42
Function of lipid rafts - prions
- rafts transmit infected cells by bumping into uninfected rafts - rafts coalesce and allow for infection - infection then propogated
43
Leukocytes
leukocytes = immune cells USUALLY - migrate to regions of infection (inflamed tissues) / antigen - makes swell more
44
Leukocyte response to vascular inflammation
- tether to endothelial cell selectins - chemokines bind to leuk and make it slow down - triggers intracellular signal that makes leukocytes adhere firmly to endothelial intercellular adhesion molecules / ICAMs and assembles lipid rafts - follow chemokines and transendothelially migrate
45
Antherosclerotic lesions and thrombotic events
- leukocytes / monocytes now sucked in form free radicals that oxidize LDLs (cholesterol) - differentiate into macrophages - collect oxidized cholesterol - become foam cells that accumulate at the site of inflammation and form plaque - plaque buildup causes occlusion by thrombosis = ischemia
46
Studying lipid rafts
Detergent-resistant membrane fraction isolation - centrifugal fractions based on density - fractionation Cholesterol depletion with cyclodextrins - hydrophobe cavity extracts cholesterol - also extracts from raft eventually - inhibit synthesis and sequestration using other chemicals Microscopy
47
Movement between leaflets
- lateral movement as per fluid mosaic model - phospholipid movement from one leaflet to another possible using enzyme flippase, but requires a lot of energy
48
3 classes of membrane proteins
Integral membrane-bound proteins - embedded into hydrophobic core --0--- - span across bilayer Lipid anchored proteins - associated with one surface of the bilayer _p__ Peripheral membrane proteins - associated with one surface of the bilayer - attached to polar membrane surface OR indirectly via other proteins ,, --0---
49
Integral membrane proteins
single pass transmembrane protein - alpha helix spans membrane multi-pass TM protein ex. bacteriorhodopsin 7-transmembrane spanning serpentine domain beta-barrels channels - porins
50
Lipid anchored proteins
acylation - lipid anchor added to n-terminus phenylation - lipid anchor added to c-terminus GPI anchor - hydrophobic anchor on c-terminus
51
Peripheral proteins
interact with another integral membrane protein = protein-protein interactions O<>{ OR lipid-binding motifs that contain amino acids allowing protein to interact with polar head groups of membranes O{ can de and re-attach

Biology 2B03 (12 decks)

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