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Biochemistry I > FINAL - CH 5 > Flashcards

FINAL - CH 5 Flashcards

1
Q

Protein Purification Factors

A
  1. pH
  2. Temperature
  3. Presence of degradative enzymes
  4. Adsorption to surfaces
  5. Long-term storage
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2
Q

Order of fractions in centrifugation

A

Nuclear
Mitochondrial
Membrane
Cytosol

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3
Q

Soilbility

A

Salting out

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4
Q

Ionic charge

A

ionic exchange chromotography
Electrophoresis
Isoelectric focusing

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5
Q

Polarity

A

Hydrophobic interaction chromotography

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6
Q

Size

A

Gel filtration chromotography

SDS-PAGE

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7
Q

Binding specificity

A

Affinity chromotography

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8
Q

Chromatography Techniques

A

Gel filtration
Ion-Exchange
Affinity

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9
Q

High Performance Liquid Chromatography

A

version of gel filtration

leads to greater separation of proteins that are a similar size

High pressure is required to force buffer and protein through the column

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10
Q

pH = pI

A

No interaction will occur between protein and column

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11
Q

pH > pI

A

-

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12
Q

pH < pI

A

+

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13
Q

Cation exchanger

A
  • charged column

CMC

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14
Q

Anion exchanger

A

+ charged column

DEAE

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15
Q

Specific activity

A

total units of activity / total protein

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16
Q

Multifold increased purity

A

Specific activity of sample in question/Specific activity of sample before it

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17
Q

Total yield***

A

total units of sample in question/Total units of step 1

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18
Q

Total multifold purification

A

Specific activity in question/Specific activity in step 1

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19
Q

Technique for the separation and visualization of
proteins based on the migration of charged
proteins in an electric field

A

Electrophoresis

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20
Q

PAGE

A

Polyacrylamide gel electrophoresis

Separates proteins on the basis of charge and size

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21
Q

percentage of gel in PAGE

A

low % - small molecules migrate faster, better separation between large molecules

High% - separation better between small molecules

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22
Q

SDS-PAGE

A

detergent that assists molecules to the anode (+)

denatures proteins

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23
Q

Coomassie Brilliant Blue G-250

A

used to stain proteins in PAGE

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24
Q

Isoelectric focusing

A

Separates proteins based on isoelectric point

Proteins will stop migrating once they reach the pH that matches their PI

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25
Q

Two Dimensional (2D) Gel Electrophoresis

A

Isoelectric focusing combined with SDS-PAGE

…………………………………High mass

………………………………….Low Mass
Low PI…………..High PI

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26
Q

2D Differential In-Gel Electrophoresis

A

Uses covalently bound fluorescent dyes (Cy3 and Cy5) to
distinguish two proteins run on the same SDS
PAGE gel

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27
Q

Edman degradation can sequence an oligopeptide up to ____ amino acid residues

A

50

28
Q

Edman degradation

A

PITC is covalently attached to the N-terminal amino acid and then treated with TFA which cleaves between the first and second amino acid

29
Q

For peptides LARGER than 50 AAs

A

enzymatic cleavage with trypsin and

chymotrypsin is performed

30
Q

chymotrypsin

A

Cleaves at C-side of Tyr, Trp, and Phe

31
Q

trypsin

A

Cleaves on the C-side of Lys and Arg

32
Q

Cyanogen bromide

A

Cleaves on C-side of Met

33
Q

V8 protease

A

Cleaves on the C-side of Asp and Glu

34
Q

Mass Spectrometry

A

Measures the mass of small peptide
fragments

mass-to-charge ratio (m/z)

35
Q

Peptide Ionization Methods

A

(mass spec)
Electrospray ionization
Matrix-assisted laser desorption ionization

36
Q

Electrospray ionization

A

(mass spec)
Solution of peptide is sprayed

high voltage forms fine, highly charged droplets from which solvent rapidly evaporates

Dry N2 gas promotes the evaporation

The charges result from the protonation of Arg and Lys.

37
Q

Matrix-assisted laser desorption ionization

A

Tryptic fragments are embedded in a light-absorbing
matrix

Fragments are released as charge molecules after laser
exposure

A detector determines the mass

38
Q

DNA sequencing

A

Faster than sequencing AA

can be used only if Gene has been isolated

39
Q

Solid Phase Peptide Synthesis

A

generate oligopeptides of up to 25 amino acids

40
Q

Protein Structure Determination

A

X-ray crystallography

NMR spec

41
Q

X-ray Crystallography two steps

A

Growing diffraction quality crystals

Determining the phases of the diffracted X-rays

42
Q

Process of X-ray Crystallography

A

Xray interacts with electrons

X-ray structure is an image of the electron density of the object

Computer graphics assist in the structure determination

43
Q

better ordered crystals

A

Have higher resolution, Lesser resolution limit

44
Q

lesser ordered crystals

A

Have lower resolution, Higher resolution limit

45
Q

Crystalline proteins assume _________ structures that they have in solution

A

nearly the same

46
Q

Because crystals are hydrated,

A

Crystalline protein is in solution, which allows is to be present in normal form

47
Q

NMR spec

A

Used to determine the relative locations of atoms in a purified protein solution

48
Q

NMR vs Xray

A

Validates X-ray data

Can determine structures of proteins that fail to crystallize

More dynamic

49
Q

Antibody proteins are produced by _____ in

the immune system.

A

B cells

50
Q

The immune response is triggered by the
presence of a foreign macromolecule, often a
protein or carbohydrate, known as an ______

A

antigen

51
Q

Each B cell makes a _____ type of antibody.

A

single

52
Q

B cells can make:

A

Two classes of Ig light chains (λ and κ)

Five classes of Ig heavy chains (µ, α, δ, ε, and γ)

53
Q

the most common immunoglobulin

A

IgG

Heavy chain: γ
Light chain: λ or κ

54
Q

IgG sub unit structure

A

γ2κ2 OR γ2λ2

55
Q

Epitope

A

Specific site on antigen that can bind to the antibody

56
Q

Each antigen is surrounded by _____ different antibodies

A

4

57
Q

Polyclonal antibodies

A

Heterogeneous mixture of immunoglobulin proteins that recognize one or more epitopes on an antigenic protein

58
Q

Monoclonal

A

Homogeneous immunoglobulin species that recognizes one epitope on an antigenic protein

59
Q

Solid step synthesis 5 steps

A

Deblocking of residue 1 attached to resin

Activation of Fmoc-blocked residue 2

coupling of AA

Final deblocking

Cleavage and deprotection

60
Q

Generation of Polyclonal Antibodies

A

Immunize rabbit 4 times of 6 week period

obtain blood sample

Blood will contain non specific and specific antigens

Purification of antigen specific antibodies using affinity chromotography

61
Q

Generation of Monoclonal Antibodies

A

Multiple immunizations of lab animal

Isolate B cells

Fuse B cells and tumor cells

both antigen specific and non specific hybridomas will grow

isolate antigen specific by screening

Clone to increase amount

62
Q

Western Blotting uses 2 antibodies

A

Primary (protein-specific)

Secondary (detection antibody)

63
Q

Western blotting is used to:

A

detect proteins separated by gel

electrophoresis

64
Q

antibody-based technique used to identify
proteins in cells that have been chemically
treated in a way that preserves cell
architecture

A

Immunofluorescence

65
Q

antibodies are used to identify proteins that are associated with protein antigens by combining mass spec

A

Immunoprecipitation

Biochemistry I (13 decks)

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