Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

BIOL 200 > KH7 > Flashcards

KH7 Flashcards

1
Q

what is chromatography

A

separation of components based on their differential interactions with an immobile (solid) material

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

describe mobile phase of chromatography

A

liquid or gas moves continuously past the solid gas phase
usually liquid - an aq buffer

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

what happens to protein molecules in chromatography

A

protein molecules are moved along in the mobile phase but at rates that depend on how much they interact with the solid phase

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

what are different chromatographic methods based on

A

different kinds of interactions of the proteins with the solid phase

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

where is protein chromatography generally done

A

in. columns with a solid phase made up of milimeter sized beads

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

name 3 types of chromatography

A

gel filtration
ion exchange
antibody affinity chromatography

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

describe gel filtration chromatography

A

separation based on size
solid phase gel beads have pores of molecular dimensions
mobile phase freely enters and exits the beads and flows between beads

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

describe gel filtration chromatography and relationship of size

A

smaller proteins can enter and exit the beads through pores
large proteins cannot enter - they do not waste time diffusing within beads - run out ahead of small proteins

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

describe ion exchange chromatography

A

separation based on electric charge
protein molecules with a net electric charge will bind to immobile phase having the opposite charge
electrostatically bound proteins can be released by flowing a salt solution through the column
(displacement of the protein by Na+ or Cl- is ion exchange)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

what is an epitope

A

antibody is a protein that recognizes by highly specific binding a molecular target = epitope - present on antigen molecule

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

how many epitopes does an antibody recognize

A

one antibody
but number of possible different antibodies and different epitopes is almost infinite

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

what can antibodies be raised against

A

any kind of chemical antigen including proteins

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

what happens when epitope is recognized by antibody

A

antibody is unique to the particular protein used as an antigen
Antibody will recognize and bind to only that individual protein - even in complex mixture of proteins

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

describe antibody affinity chromatography

A

antibodies specific for any particular protein can be covalently coupled to the solid phase
if complex protein mixture is flowed through column then only protein to which antibody binds to will be retained
all other proteins can be washed away
target protein can be released from antibody by lowering pH (partial denaturation)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

describe primary and secondary antibodies

A

primary recognizes the epitope of interest
secondary recognized the primary antibody

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

what is CDR (antibodies)

A

Complementarity Determining Regions
varies among antibodies within a species
region of primary antibody that recognizes epitope of interest

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

what is constant region (antibodies)

A

constant among antibodies within a species but different from species to species
region of primary antibody that is recognized by secondary antibody from another species

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

what is immunoblot (western blot)

A

using antibodies to recognize individual protein species in a complex mixture of proteins separated by SDS PAGE

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

name and describe steps of general method of immunoblotting

A

1 - electrophoresis and transfer (turn current on)
2 & 3 - antibody detection (add antibodies, then add 2nd antibodies - will bind to constant region and add a way to make it visible)
4 - chromogenic detection (detect single bond that corresponds to the band recognized by the primary antibody - indirect immuno detection)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

describe indirect immuno detection of immunoblot

A

sandwich immunodetection
primary antibody against the antigen
secondary antibody against the primary antibody

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

what is convenient about immunoblot

A

commercial supply is enzyme linked 2nd antibody - as universal reagent
usually buy 2nd antibody

22
Q

describe what immunoblot shows

A

generic stain is used to show all proteins
able to look at all the proteins and their amounts

23
Q

what is immunoprecipitation and co-immunoprecipitation - generally

A

a protein complex can be isolated from a protein mixture by using an antibody that is specific for one protein of the complex

24
Q

what is immunoprecipitation and co-immunoprecipitation - specifically

A

recovery of protein complexes that bind to a specific antibody
the antibody:antigen complex is recovered by precipitation
immunoprecipiate will contain the protein carrying the epitope recognized b y antibody and any partner proteins stably associated with that protein - (this is co immunoprecipitation - co-IP)

25
Q

explain an example western blot/co-ip situation

A

western blot of SDS solubilized cells reveals presence of glucocorticoid receptor GR and PPAR alpha in cells treated (+) or untreated (-) with GR ligand
then COIP experiment

26
Q

describe COIP experiment with GR and PPAR alpha - 3 steps

A

1 - non sds cell homogenate (natural, non denatured proteins, bound to partners)
2 - immunoprecipitate wuth anti GR antibody
3 - add SDS and do westen blot looking for presence of PPAR alpha in immunoprecipitate

27
Q

describe conclusions of GR - PPAR alpha coip

A

PPARalpha forms a stable complex with GR but only when GR is bound to its ligand

28
Q

describe immunoflorescence microscopy

A

studying distribution of proteins at the cellular and subcellular level using antibodies that specifically recognize the protein of interest

29
Q

describe how to do indirect immunofluorescence

A

involves antibodies raised against constant region of antibodies

30
Q

how to make antibodies raised against constant region of antibodies (immunoflorescence microscopy)

A

inject purified mouse antibodies into rabbit
rabbit will recognize mouse antibodies as foreign and make antibodies against mouse antibodies

31
Q

what permits detection in fluorescence microscope

A

chemical coupling of a fluorochrome to the secondary antibody

32
Q

describe example of immunoflorescence

A

detection of the glucose transporter GLUT2 (yellow green) in a section of rat intestinal wall
GLUT2 present on lateral and basal plasma memebrane surfaces but not at apical surface (brush border)

33
Q

what is double label fluorescence

A

to simultaneously visualize 2 proteins

34
Q

describe double label fluorescence

A

target different proteins
primary = one on mouse and one on rabbit
secondary = anti mouse antibody and anti rabbit antibody
must come from different species- way of detecting both proteins, must use different species

35
Q

two colour immunofluorescence can study what

A

study distribution of 2 proteins
vinculin (red) and actin (green) within a single cell

36
Q

describe how two colour immunofluorescence works for vinculin

A

primary antibody (from species A) raised against vinculin
secondary antibody (red fluorescence) raised against antibodies of species A

37
Q

describe how two colour immunofluorescence works for actin

A

primary antibody (from species C) raised against actin
secondary antibody (green fluorescence) raised in species D (or B) against antibodies of species C

38
Q

what is GFP

A

green fluorescent protein
major advancement
jellyfish protein that fluoresces green

39
Q

describe what happens when genes encoding GFP are introduced into cell of organism

A

fluorescent GFP is produced in living cells

40
Q

what can GFP be used for in cells of organisms - 2 ways

A

used as reporter gene for transcriptional control elements
made into fusion proteins to study intracellular protein localization

41
Q

describe GFP as a reporter gene to reveal gene promoter driven transcription patterns

A

substituted GFP for reporter gene
so will produce GFP for what cell is actively doing (what gene is coding for)

42
Q

describe GFP fusion proteins to study intracellular protein localization

A

tagged at end of protein
done at DNA level
so mRNA will have GFP on it

43
Q

what does use of recombinant GFP fusion construct

A

visualize gene expressions and protein localization in living cells/organisms

44
Q

how does GFP work

A

single polypeptide chain that contains enzymatic activity that modifies some of its own aa side chains to generate fluorochrome
excited by energetic (blue light)
modified to be green

45
Q

what are the 2 thing that can happen when using recombinant GFP

A

GFP coding sequence replaces the protein coding sequence of ODR10 gene
GFP coding sequence fused to protein coding sequence of ORD10

46
Q

what is ODR10

A

odorant receptor expressed only in sensory neurons and specifically targeted to tips of those neurons

47
Q

describe result of GFP coding sequence replacing the protein coding sequence of ODR10 gene

A

GFP expressed only in cells where ODR10 gene is transcriptionally active - specific sensory neurons

48
Q

describe results of GFP coding sequence fused to protein coding sequence of ORD10

A

ODR10 GFP fusion protein is targeted to the sub cellular region to which ODR10 is normally targeted

49
Q

where does fusion protein localize to

A

fusion protein localizes to nuclei (expected for transcription factor)

50
Q

what is prof KH researching

A

Ciona embryo with recombinant gene encoding SNAP 190-GFP fusion protein

BIOL 200 (29 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions