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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
Two Dimensional (2D) Gel Electrophoresis
Isoelectric focusing combined with SDS-PAGE .......................................High mass ........................................Low Mass Low PI..............High PI
26
2D Differential In-Gel Electrophoresis
Uses covalently bound fluorescent dyes (Cy3 and Cy5) to distinguish two proteins run on the same SDS PAGE gel
27
Edman degradation can sequence an oligopeptide up to ____ amino acid residues
50
28
Edman degradation
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
For peptides LARGER than 50 AAs
enzymatic cleavage with trypsin and | chymotrypsin is performed
30
chymotrypsin
Cleaves at C-side of Tyr, Trp, and Phe
31
trypsin
Cleaves on the C-side of Lys and Arg
32
Cyanogen bromide
Cleaves on C-side of Met
33
V8 protease
Cleaves on the C-side of Asp and Glu
34
Mass Spectrometry
Measures the mass of small peptide fragments mass-to-charge ratio (m/z)
35
Peptide Ionization Methods
(mass spec) Electrospray ionization Matrix-assisted laser desorption ionization
36
Electrospray ionization
(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
Matrix-assisted laser desorption ionization
Tryptic fragments are embedded in a light-absorbing matrix Fragments are released as charge molecules after laser exposure A detector determines the mass
38
DNA sequencing
Faster than sequencing AA can be used only if Gene has been isolated
39
Solid Phase Peptide Synthesis
generate oligopeptides of up to 25 amino acids
40
Protein Structure Determination
X-ray crystallography | NMR spec
41
X-ray Crystallography two steps
Growing diffraction quality crystals Determining the phases of the diffracted X-rays
42
Process of X-ray Crystallography
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
better ordered crystals
Have higher resolution, Lesser resolution limit
44
lesser ordered crystals
Have lower resolution, Higher resolution limit
45
Crystalline proteins assume _________ structures that they have in solution
nearly the same
46
Because crystals are hydrated,
Crystalline protein is in solution, which allows is to be present in normal form
47
NMR spec
Used to determine the relative locations of atoms in a purified protein solution
48
NMR vs Xray
Validates X-ray data Can determine structures of proteins that fail to crystallize More dynamic
49
Antibody proteins are produced by _____ in | the immune system.
B cells
50
The immune response is triggered by the presence of a foreign macromolecule, often a protein or carbohydrate, known as an ______
antigen
51
Each B cell makes a _____ type of antibody.
single
52
B cells can make:
Two classes of Ig light chains (λ and κ) Five classes of Ig heavy chains (µ, α, δ, ε, and γ)
53
the most common immunoglobulin
IgG Heavy chain: γ Light chain: λ or κ
54
IgG sub unit structure
γ2κ2 OR γ2λ2
55
Epitope
Specific site on antigen that can bind to the antibody
56
Each antigen is surrounded by _____ different antibodies
4
57
Polyclonal antibodies
Heterogeneous mixture of immunoglobulin proteins that recognize one or more epitopes on an antigenic protein
58
Monoclonal
Homogeneous immunoglobulin species that recognizes one epitope on an antigenic protein
59
Solid step synthesis 5 steps
Deblocking of residue 1 attached to resin Activation of Fmoc-blocked residue 2 coupling of AA Final deblocking Cleavage and deprotection
60
Generation of Polyclonal Antibodies
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
Generation of Monoclonal Antibodies
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
Western Blotting uses 2 antibodies
Primary (protein-specific) Secondary (detection antibody)
63
Western blotting is used to:
detect proteins separated by gel | electrophoresis
64
antibody-based technique used to identify proteins in cells that have been chemically treated in a way that preserves cell architecture
Immunofluorescence
65
antibodies are used to identify proteins that are associated with protein antigens by combining mass spec
Immunoprecipitation

Biochemistry I (13 decks)

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