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Biochemistry I > Chapter 5 - Methods in Protein Biochem > Flashcards

Chapter 5 - Methods in Protein Biochem Flashcards

1
Q

If pH > PI:

A

Molecule is negative and will bind to positively charged medium

Called an anion exchanger

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2
Q

If pH < PI:

A

Molecule is positive and will bind to negatively charged media

Called a cation exchanger

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3
Q

1,000 g for 5 minutes

A

Supernatant: Mitochondrial, membrane, cytosol
Pellet: Nuclear fraction

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4
Q

10,000 g for 10 minutes

A

Supernatant: Membrane, cytosol
Pellet: Mitochondrial

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5
Q

15,000 g for 15 minutes

A

Supernatant: Cytosol
Pellet: Membrane

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6
Q

extracting a protein bound to a column by a solvent that removes the protein from the stationary phase to the mobile phase

A

Elution

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7
Q

Generally, the chemistry of Fmoc blocking is straightforward for most amino acids during solid state peptide synthesis. There is one amino acid, however, that presents a problem for Fmoc blocking during solid state peptide synthesis. That amino acid is

A

Lysine

It has an R-NH3+ group that will react with Fmoc and thus this side chain group also needs blocking

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8
Q

this method uses an X-ray beam aimed at a homogeneous protein crystal in solid phase.

A

X-ray crystallography

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9
Q

Provides information on the structure of a purified protein based on its nuclear spin properties in a magnetic field

A

NMR Spectroscopy

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10
Q

Use of high-affinity antibodies linked to a dense carbohydrate bead to isolate protein antigens

A

Immunoprecipitation

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11
Q

Low-percentage gel

A

Increases rate of protein migration

Separates larger proteins at expense of smaller protein separation

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12
Q

High-percentage gel

A

Decreases rate of protein migration

Separates smaller proteins at expense of larger protein separation

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13
Q

2-D gels:

A

_________________Larger

_________________Smaller
Low PI High PI

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14
Q

_______ cleaves on the carboxy side of aromatic amino acids

A

Chymotrypsin

W, Y, F

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15
Q

_______ cleaves Lysine

A

Trypsin

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16
Q

Luciferase

A

An enzyme found in bioluminescent
organisms

Uses luciferin as a substrate

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17
Q

If you add luciferin substrate to different protein samples:

A

The protein sample that illuminates contains luciferase

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18
Q

membrane bound cells can be homogenized in one of three ways:

A

Sonication

Shearing (with French press)

Incubation with mild detergents

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19
Q

Centrifugation

A

Performed to increase the concentration

and purity of the target protein in the sample

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20
Q

Cell suspensions are prepared by

A

mincing tissue mechanically or enzymatically to generate membrane bound cells

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21
Q

Specific activity

A

The total amount or activity of the target protein divided by the total amount of protein in the fraction

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22
Q

How to find the magnitude of purification

A

Multiply the specific activities of cell extract (un purified sample) and cytosol fraction

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23
Q

Protein Purification Factors

A 
pH
Temperature
Presence of degradative enzymes
Adsorption to surfaces
Long-term storage
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24
Q

Protein characteristic: Solubility

A

Salting out

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25
Q

Protein characteristic: Ionic charge

A

Ionic exchange solubility
Electrophoresis
Isoelectric focusing

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26
Q

Protein characteristic: Polarity

A

Hydrophobic interaction chromatography

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27
Q

Protein characteristic: Size

A

Gel filtration chromatography

SDS-page

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28
Q

Protein characteristic: Binding specificity

A

Affinity chromatography

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29
Q

Salting out

A

causes the formation of insoluble protein
aggregates that are functional when
resolubilized

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30
Q

Process of salting out

A

a) Salt is added to a concentration just below the precipitation point of the target protein
b) After centrifugation, unwanted proteins are discarded and more salt is added to salt out the target protein
c) After a second centrifugation, the protein is recovered as a precipitate.

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31
Q

As you add more salt:

A

Salt and ions are competing for solvent

Salt wins staying in solution forcing other proteins into pellet

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32
Q

Dialysis

A

Used to remove ammonium sulfate from protein sample

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33
Q

Gel Filtration Chromatography separates proteins based on:

A

Size

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34
Q

In Gel Filtration Chromatography:

A

Large travel faster than small proteins

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35
Q

High Performance Liquid Chromatography (HPLC)

A

Leads to greater separation of proteins similar in size

Uses pressure to push buffer and molecules into column

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36
Q

Positively charged, anion exchange matrix

A

(DEAE)

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37
Q

Negatively charged, cation exchange matrix

A

(CMC)

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38
Q

Ion exchange chromatography

A

Separates molecules on the basis of differences in

their net surface charge

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39
Q

Affinity Chromatography

A

Exploits specific binding properties of the target
protein to separate it from other cellular proteins
that lack this function

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40
Q

High-affinity ligand for the target protein is

________ to the matrix bead.

A

covalently linked

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41
Q

The binding between the ligand and target
molecule must be _____ to allow target
molecules to be ______ in an active form.

A

reversible

removed

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42
Q

The specific activity is a measure of ______: it increases during purification of an enzyme and becomes _______ when the enzyme is pure.

A

enzyme purity

maximal and constant

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43
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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44
Q

Electrophoresis is carried out:

A

In gels made up of the cross-linked

polymer polyacrylamide

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45
Q

PAGE

A

Polyacrylamide gel electrophoresis

Separates proteins on the basis of charge and size

Percentage of gel is important depending on protein
size

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46
Q

SDS-PAGE

A

Uses sodium dodecyl sulfate, a detergent
that adds a net negative charge to the protein to aid in
migration to the anode

Used to denature the proteins

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47
Q

Migration from _____ to _____

A

Cathode (-)

Anode (+)

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48
Q

Staining of proteins in electrophoresis:

A

Coomassie Brilliant Blue G-250

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49
Q

Isoelectric Focusing

A

Separates proteins based on isoelectric point

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50
Q

Two Dimensional (2D) Gel Electrophoresis

A

Isoelectric focusing combined with SDS-PAGE

Separates proteins based on pI and
molecular mass

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51
Q

2D Differential In-Gel Electrophoresis (DIGE)

A

Proteins are covalently labeled with different
fluorescent dyes

Uses Cy3 (green) and Cy5 (red)

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52
Q

Nobel prize in Chemistry for protein sequencing

A

Frederick Sanger

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53
Q

Improved Sanger’s protein sequencing

method

A

Edman degradation

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54
Q

Edman degradation:

________ (PITC) is covalently
attached to:

A

Phenylisothiocynate

the N-terminal amino acid andthen treated with TFA

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55
Q

Edman degradation can

sequence an oligopeptide up to:

A

50 amino acid residues

56
Q

For proteins longer than 50 AA:

A

enzymatic cleavage with trypsin and

chymotrypsin is performed

57
Q

Trypsin:

A

Cleaves on the C-side of Lys and Arg

58
Q

Chymotrypsin:

A

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

59
Q

Cyanogen bromide:

A

Cleaves on C-side of Met

60
Q

S. aureus:

A

Cleaves on the C-side of Asp and Glu

61
Q

Sanger reaction can determine ______ in protein sequencing

A

N-terminus AA

62
Q

Protein Sequencing: How to determine the composition

A
  1. Purify the protein of interest – separate away from all
    other types of proteins and biomolecules
  2. Estimate the molecular weight of the protein
  3. Establish the composition by complete hydrolysis of the protein under acidic conditions
63
Q

Protein Sequencing: How to determine the order

A
  1. Determine the C-terminal amino acid
  2. Identify the N-terminal amino acid
  3. Cleave disulfide bonds between and within polypeptides
  4. Cleave peptide bond using proteases in a specific way.
  5. Determine the aa sequences through repeated cycles of Edman degradation.
  6. Conduct a second round of protein cleavage and compare the aa sequences of the overlapping sets of peptide fragments.
  7. Finally determine the positions (if any) of the disulfide bonds.
64
Q

How to determine the C-terminal amino acid

A

Use carboxypeptidase – enzyme that removes the last (C-terminal) amino acid in a free form by breaking the peptide bond

Hydrolyzes the peptide bond nearest the C-terminus

65
Q

How to identify the N-terminal amino acid

A

Use 5-dimethylamino-1-naphthalenesulfonyl chloride (dansyl chloride)-fluorescent compound that reacts with primary amines

66
Q

How to cleave disulfide bonds between and within polypeptides

A

Use 2-mercaptoethanol or another mercaptan

Treat with iodoacetate to prevent the reformation of disulfide bonds through oxidation by O2

67
Q

Cleave peptide bond using proteases in a specific way

A

Trypsin – Cleaves on the C-terminal side of Lys and Arg residues

Chymotrypsin – Cleaves on the C-terminal side of Tyr, Phe, and Trp

Chemical proteases also can cleave proteins: Cyanogen Bromide (CNBr) – cleaves on the C-terminal side of Met

68
Q

How to determine the aa sequences through repeated cycles of Edman degradation.

A

If the sequence of each peptide is determined, the entire protein sequence can be reassembled from the fragments

69
Q

Mass Spectrometry

A

Measures the mass of small peptide fragments

Measures the mass-to-charge ratio (m/z)

Can be used to determine the molecular mass

70
Q

Peptide Ionization Methods

A

Electrospray ionization (ESI)

Matrix-assisted laser desorption ionization (MALDI)

71
Q

Electrospray ionization (ESI)

A

Solution of peptide is sprayed from a narrow capillary tube maintained at high voltage (~4000 V), forming 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.

72
Q

Matrix-assisted laser desorption ionization (MALDI)

A

Tryptic fragments are embedded in a light-absorbing
matrix.

Fragments are released as charge molecules after laser
exposure.

A detector determines the mass.

73
Q

When the gene is isolated, sequencing the DNA can be:

A

Faster and more accurate than sequencing the protein.

74
Q

Most proteins are now sequenced in this indirect way:

A

DNA Sequencing

75
Q

If the gene has not been isolated, direct sequencing of _____ is necessary.

A

peptides

76
Q

Amino acid sequence provides information about protein structure that is not revealed by DNA sequencing:

A

Location of disulfide bonds

77
Q

Solid Phase Peptide Synthesis

A

a method to generate oligopeptides of up to

25 amino acids.

78
Q

Solid Phase Peptide Synthesis involves adding ______ to the peptide through a ______ on the amino terminus

A

one amino acid at a time

covalent linkage

79
Q

Used to synthesize peptide antigens for antibody
production and to manufacture peptide-based
therapeutic drugs to treat a variety of diseases.

A

Solid Phase Peptide Synthesis

80
Q

Solid Phase Peptide Synthesis Step 1

A

De-blocking of residue 1 attached to resin

81
Q

Solid Phase Peptide Synthesis Step 2

A

Activation of Fmoc-blocked residue 2

82
Q

Solid Phase Peptide Synthesis Step 3

A

Coupling of AAs

83
Q

Solid Phase Peptide Synthesis Step 4

A

Final de-blocking of peptide with 20 AAs

84
Q

Solid Phase Peptide Synthesis Step 5

A

Cleavage form resin and de-protection of all amino side chains

85
Q

Two primary methods for determining molecular

structure:

A

X-ray crystallography - use crystal of pure protein

NMR Spectroscopy - measures magnetic characteristics of each atom

86
Q

X-ray crystallography

A

Based on the diffraction of X-rays by protein

crystals

87
Q

X-ray crystallography 2 steps:

A

Growing diffraction quality crystals

Determining the phases of the diffracted X-rays

88
Q

laboratory X-ray generators

A

Synchrotrons

89
Q

X-ray crystallography determined structure of ____

A

Myoglobin

90
Q

Protein crystals are highly _______

A

hydrated

40-60% water by volume

91
Q

Protein crystals typically have resolution limits in the

range:

A

1.5 to 3.0 Å

92
Q

Better ordered crystals have _____ resolution and _____ resolution limit

A

higher, lesser

93
Q

Less ordered crystals have _____ resolution and _____ resolution limit

A

lower, higher

94
Q

Many ____ are catalytically _____ in the crystalline

state.

A

enzymes

active

95
Q

Crystal packing forces ____ greatly perturb the

______ of protein molecules.

A

do not

structures

96
Q

Protein in a crystal is essentially ______ because it is
bathed by _______ over its surface
except for the few

A

in solution

solvent of crystallization

97
Q

NMR Spec only works for proteins less than:

A

100 kDa

98
Q

NMR Spec Validates:

A

X-ray data

99
Q

NMR spec can determine:

A

Structures of proteins that fail to crystallize

100
Q

NMR spec resolution:

A

2-2.5 Å resolution

101
Q

NMR Spectroscopy

A

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

102
Q

Antigen

A

a foreign macromolecule, often a protein or carbohydrate, that triggers the immune system

103
Q

Antibody proteins are produced by ____ in

the immune system.

A

B cells

104
Q

B cells can make:

A

Two classes of Ig (immunoglobulin) light chains (λ and κ)

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

105
Q

IgG

A

the most common immunoglobulin, is equally distributed between the blood and the extravascular fluid.

106
Q

IgG structure:

A

Light chain: λ or κ
Heavy chain: γ
Subunit structure: γ2λ 2 OR γ2κ2
Molecular mass: 150 kD

107
Q

The association between
antibodies and their antigens
involves:

A

van der Waals, hydrophobic, hydrogen bonding, and ionic

interactions.

108
Q

Antigen-antibody complex have:

A

high specificity and strength

109
Q

The immune system has the potential to produce

an enormous number of different antibodies

A

> 10^18

110
Q

The diversity in antibodies results from

A

gene recombination and mutation

111
Q

Polyclonal antibodies

A

Heterogeneous mixture of immunoglobulin proteins

recognize one or more epitopes on an antigenic protein

112
Q

Monoclonal antibodies

A

Homogeneous immunoglobulin species

recognizes one epitope on an antigenic protein

113
Q

Western blotting

A

Used to detect proteins separated by gel electrophoresis

114
Q

Western blotting uses two antibodies:

A

Primary (protein-specific)

Secondary (detection antibody)

115
Q

Protein-coding sequences of highly antigenic
peptides are added to protein-coding
sequences of cloned genes.

A

Epitope Tagging

116
Q

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

A

Immunofluorescence

117
Q

Enzyme Linked Immunosorbent

Assay (ELISA)

A

Identifies low level antigenic proteins

Typically used in biological samples

The amount of substrate converted indicates the amount of protein present

118
Q

Immunoprecipitation

A

Variation of affinity purification

119
Q

In in mmunoprecipitation _______ is ______ linked to a carbohydrate bead.

A

monoclonal antibody

covalently

120
Q

In immunoprecipitation the ________ combination is

used to _______ from other proteins physically using _________

A

antibody-bead

separate protein antigens

low speed centrifugation

121
Q

In immunoprecipitation antibodies can be used to identify proteins that are associated with __________________ by combining _____________

A

protein antigens in large cellular complexes

immunoprecipitation with mass spectrometry

122
Q

addition of a phosphate group makes the protein more:

A

negatively charged

123
Q

chelation affinity chromatography

A

purifies recombinant proteins with 6 or more histidine residues which have strong affinity for divalent metals

124
Q

Antibody column

A

isolates antigenic proteins in the sample

125
Q

Epitope tag

A

protein-coding sequences of highly antigenic peptides

126
Q

ESI

A

electrospray ionization (mass spec)

127
Q

MALDI

A

matrix-assisted laser desorption/ionization (mass spec)

128
Q

ESI uses

A

high voltage

generates highly charged molecule in gas phase

129
Q

MALDI uses

A

laser

proteins are fragmented and charged

130
Q

Primary antibody

A

facilitate antigen-antibody interaction

131
Q

Secondary antibody

A

recognizes primary antibody, assists in detection

132
Q

Each B cell makes how many antibody type

A

One

133
Q

How many classes of immunoglobulin heavy chain are produced

A

5

134
Q

How many classes of immunoglobulin light chain are produced

A

2

135
Q

Secondary antibodies are typically from:

A

A different animal as the primary antibody

Biochemistry I (13 decks)

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