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MT1 Flashcards

0
Q

proto-oncogenes

A

accelerate growth

mutations dominant - increase activity

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1
Q

tumor supressor gene

A

prevents cell proliferation

mutation –> recessive –> must rid both copies

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2
Q

testing necessity

A

remove gene –> process doesn’t occur

it was necessary for the process

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3
Q

testing sufficiency

A 
geneX + test tube --> process occurs
having geneX (and other things) was sufficient for process
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4
Q

light and heavy chains are connected by

A

disulfide bonds

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5
Q

names of two regions of antibodies

A

Fab, Fc (constant)

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6
Q

antigen

A

protein/small molecule that antibodies bind to

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7
Q

Western Blots:

answers what question(s)?

A

protein of interest: present? amount? molecular weight?

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8
Q

Western Blots:

procedure

A
  1. Harvest protein: Lyse cells
  2. SDS-PAGE + coomassie blue stain
  3. Transfer to membrane
  4. probe with antibody to light up single band
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9
Q

Immunoprecipitaion

-purpose

A

purify protein (with antibodies)

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10
Q

Immunoprecipitation: procedure

A
  1. Harvest cells : lyse with Dounce homogenizer + mild detergent
  2. Clear lysate: centrifuge
  3. Add Ab to supernatant; toss pellet
  4. Purify Ab: Staph protein A binds to Fc region of IgG -> centrifuge out
  5. SDS + Western blots
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11
Q

protein A

A

Staph protein that binds Fc region of IgG

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12
Q

Rituxan

A

‘humanized’ mouse Ab: Fc from human, Fab from mouse
binds CD20 B cells : binds all B cells for destruction; healthy grow back
(for Non-Hodgkins B cell lymphoma)

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13
Q

Subcellular fractionation

purpose

A

isolate protein/organelle of interest

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14
Q

Subcellular Fractionation

Procedure

A
  1. lyse cells with Dounce Homogenizer
  2. 1G - nuclei pellet
  3. 12G - pellet mito, lysosomes, ribosomes
  4. 50G - pellet ER, Golgi, Plasma; supernatant cytosol
  5. 65G on sucrose - separate last pellet
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15
Q

microscope condenser

A

focuses light

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16
Q

compound objective lens

A

objective bends and magnifies light

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17
Q

limit of resolution
eq
value

A

distance btween 2 objects that can be resolved
d = .61(lambda)/nsin(a)
n = refractive index between sample + objective (eye) air(1), oil (1.5)
a = angle

value = 72degrees -> 200nm

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18
Q

other name for nsin(a)

A

NA - numerical aperature - 1.4 typically

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19
Q

Immunofluorescence

role of formaldehyde, methanol

A

formaldehyde crosslinkjs, methanol precipitates

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20
Q

Immunofluorescence

A
  1. fix cells: cross link () and precipitate protein ( )
  2. Primary antibody: bind protein of interest
  3. Secondary antibod + fluorophore: recognizes Fc of the above
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21
Q

What is the limit of immunofluorescence

A

need antibody

fix cells - no dynamics

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22
Q

Electron Microscope

theoretical and practical limits

A

theor (.3 nm)

practical - 1 nm

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23
Q

Electron microscopy

focus electrons by

A

magnets - hi voltage thru vacuum

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24
Q

Electron Microscopy

-cons

A

need thin slices
heavy fixation
low contrast -> heavy staining
DAMAGING

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25
Q

Membrane Function

A
  1. concentrate molecules, make gradients
  2. control exchange
  3. communication
  4. compartmentalize cell + organelles (processes)
  5. shape / adherence to surface
  6. 2d–> 3D
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26
Q

First membrane characterization

A

more lipid soluble dyes –> went into cells better

therefore made of lipids

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27
Q

Determination of membrane composition

A
  1. use RBC: bc easy/cheap harvest, no organelle (membranes), exist as single cells of uniform size
  2. ghosts: hypotonic conditions –> lyse and cyto leaks out
  3. Langmuir trough to measure SA

SAfound = 2xSAnorm
therefore, bilayer

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28
Q

headgroups of phosphoglycerides

A 
phosphotidyl:
ethanolamine
choline
inositol
serine
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29
Q

fatty acids #carbons

A

14-25

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30
Q

phosphoglyceride structure

A 
R(headgroup)
-OP=O
    O
    C-C-C glycerol
        O  OC=O
   O=C  R
        R
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31
Q

sphingolipids

A

longer than phospholipids

form ‘raft’ cluster of thicker membrane

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32
Q

cholesterol

A

small OH sterol
not in plants, rare in bacteria
occupy spaces left by unsaturated–> less fluid/permeale
higher mp

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33
Q

where do cells tend to put sugars on lipids?

A

outside leaflet

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34
Q

Important properties of lipids

A

self assembly- reseal quickly

3. fluidity - lateral diffusion between lipid, spread

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35
Q

Flipases

A

flip phosphoglycerides to keep 2 leaflets distinct

36
Q

FRAP
purpose
procedure

A

study protein dynamics:
Fluorescent recovery after photobleaching
1. strong laser excitation –> bleach GFP
2. watch for green recovery - proportional to how dynamicds

37
Q

Types of Membrane protein

A
  1. hydrophobic a helix: 20 aa & 6-7 turns
  2. multi-pass: up to 20 times: often bundle pore with polar interior (helix wheel diagrams)
  3. beta barrels
  4. lipid modified : post translational lipid addition to protein
  5. peripheral membrane: entirely outside - attach with non-covalent **removed with hi salt
  6. amphipathic helix : one side polar, hphobic
38
Q

TritonX-100

A

nonionic detergent - hphobic tail, polar head

preserve enzymatic activity of membrane protein etc

39
Q

Trypsin

A

enzyme degrades proteins but can’t cross membrane

40
Q

Dounce homogenizer

A

breaks cell without disrupting membranes/vesicles

41
Q

how to harvest peripheral protein

A

hi salt + (Dounce)

42
Q

how to harvest luminal protein (in cell)

A

pH 11: proteins leak out

43
Q

how to harvest integral membrane protein

A

extract with detergent (tritonX100?)

44
Q

what would naturally happen if there were no Na/K pumps

A

negatively charged protein/sugars/aa’s attract Na+ from exteria
water moves with Na
lysis

pump salt / cell wall / pump water

45
Q

ions in cell: where are they higher?

A

inside - K+

outside - Na+ Ca2+(signaling) Cl- (counterion)

46
Q

gated ion channels

A

specific for partic ion

cell controls with open/closed bc BIDIRECTIONAL

47
Q

Transporters

A

large polar molecules down gradient

passive

48
Q

tunicamycin

A

blocks oligosaccharyl transferase

49
Q

Na/K+ pump

A

Active - Uses ATP to drive rxn
2 K+ into cytoplasm
3 Na outside
hydrolyze ATP in cytoplasm

50
Q

Sucrose/H+ active transport

A

symporter
sucrose into cell
H+ into cell

51
Q

the inside of the cell is (acidic / basic)

A

basic - hi pH

52
Q

Na+ / H+ active transport

A

antiporter in plants
H+ into cytosol (basic)/ from vacuole (acidic)
Na+ into vacuole (Hi)

**vacuole conditions like exterior conditoins **

53
Q

cholera toxin

A

secretion of Cl- into gut –> h2o follows

cure: add Na+ glucose to water
- > Na+gluc transporter takes over and brings Na into cell -> water follows

54
Q

Roles of ECM

A

scaffold tissue/organs
separate tissues with basement membranes (not lipidy though mems)
pathway for migration events

55
Q

components of ECM

A

collagen
proteoglycans
multi-adhesive matrix proteins
integrins?

56
Q

collagin

A

hi tensile strength
makes helical bundles
14 types

57
Q

proteoglycans

A

protein + polysac

make cartillage

58
Q

multi-adhesive matrix protein

types

A

laminin, fibronectin, nidogen

crosslink collagens + proteoglycans

59
Q

matrix metalloproteases

A

degrade local ECM/membrane

recruit blood vessels

60
Q

integrins

A

integral membrane protein with alpha chain and beta chain (many combos)

extracellular domains of intregrins bind ECM
communicate with cytoskeleton as well

how cells communicate to ECM

61
Q

Secretory pathway overview

A

secrete proteins in right lication/orientation

dynamic - easy to reorganize

62
Q

chases

A 
(min) 
0 ER
7 Golgi
37 secretory vesicles
117 secreted from cell
63
Q

VSVG

A

Temp sensitive viral gene mutation:

32 normal 40 celsius - misfolded and stuck in ER

64
Q

new age pulse-chase experiment

A

VSVG-GFP:
Infect cells at 40degrees –> move to 32 degrees
stop at same place –> go!
virus successfully infects, but this protein is derpy

65
Q

secretion steps

A

make protein at ER membrane
modify and q control
vesicle transport

66
Q

anterograde/retrograde
else (address)
inside ER/golgi/vesicle = outside cell

67
Q

translocation

A

insert proteins into ER

68
Q

GEF

A

GTP exchange factor : switches GDP and GTP

69
Q

GAP

A

GTPase Activating Protein

timer

70
Q

SRP:G_P has low affinity for SR

71
Q

GEF#1 in translocation

A

SP + ribosome

72
Q

GEF #2 in translocation

A

SRP/ribosome/SP

73
Q

G protein

A

GTP binding protein whose activity/affinity is shifted by GAP/GEF

74
Q

SR-G_P and SRP-G_P bind each other tightly

75
Q

translocation GAP ‘Timer’

A

translocon

GAP for SRP, SR

76
Q

HSP20 family

A

of BiP chaperones in ER

77
Q

enzyme that cleaves N terminal signal peptide (NOT middle - those shouldnt be cleaved)

A

signal peptidase

dont recognize 20 hphobic transmembrane aa’s either

78
Q

translocon charge

A

hphobic middle

- charge on cytoplasmic face to attract some short + peptide ends

79
Q

Transmembrane protein

Type I

A

single pass with N term in ER/outside cell
had N term signal peptide that was recognized and inserted into ER
internal stop signal (20 hphobic residues)

80
Q

Transmembrane protein

Type II

A

single pass with N term in cytoplasm

no Nterm signal, internal start transfer, no stop transfer

81
Q

Transmembrane protein

Type III

A

single pass N term in ER/lumen
N n term signal peptide but small ++ N term, internal signal to enter (?)
post translational threading thru

82
Q

first precursor to N linked glycosylation

A

2-NAG-5-mannose

83
Q

glycosylation : what links cytoplasmic sugars to ER

A

dolichol phosphate (2P membrane spanning)

84
Q

glycosylation: sugars added inside ER

A

2NAG 9 mannose 3 glucose

85
Q

oligosaccharyl transferase

A

transfers 2 NAG 9 mannose 3 glucose inside ER

86
Q

calnexin

A

chaperone that removes last glucose if folded properly

87
Q

reticulocyte lysates

A

ribosomes, tRNA, amino acids, translation machinery

Cell Biology (1 decks)

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