Brainscape's Knowledge GenomeTM

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


See full index

Chem 193 Biochemistry C3 > PROTEIN SEPARATION AND PURIFICATION > Flashcards

PROTEIN SEPARATION AND PURIFICATION Flashcards

You may prefer our related Brainscape-certified flashcards:
AP® Chemistry flashcards Periodic Table flashcards Chemistry 101 flashcards Organic Chemistry 101 flashcards
1
Q

breaking
cells and solubilize
proteins by using
homogenizer,
grinder or sonicator

A

Homogenization of
tissue or microbial
cells to produce cell
extracts

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

use of highspeed centrifugation to prepare
subcellular fractions or to isolate specific
organelles.

A

Differential Centrifugation

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

how many subcellular fractions are obtain in differential centrifugation

A

4

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

refers to the purity of the
protein and is defined
by the ratio of activity
units to total protein.

A

Specific gravity

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

Formula of specific gravity

A

total unit of activity / total protein

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

addition of increasing amounts of a saturated
(NH4)2SO4 to the protein
solution. This method selectively
precipitates some proteins while others
remain in solution.

A

Fractionation by Salting Outs

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

salt used in fractionation

A

ammonium sulfate

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

a procedure that separates proteins from solvents (or
ammonium sulfate) by taking
advantage of the
proteins’ large size

A

Dialysis

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

separates proteins on
the basis of differential physical or
chemical interactions with a solid gel
matrix.

A

Separation and Purification by Column
Chromatography

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

Types of Column
Chromatography

A
  1. Size-exclusion chromatography/gel
    filtration chromatography
  2. Affinity Chromatography
  3. Ion-exchange chromatography
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

As the proteins flow through the column, the
proteins that interact poorly with the matrix
are eluted first from the column and can be
separated away from other proteins, which
elute more slowly

A

Column
Chromatography

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

Separates proteins according to
size.

A

Size-exclusion/gel filtration
chromatography

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

what is eluted first in Size-exclusion/gel filtration
chromatography

A

Larger proteins

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

The matrix or stationary phase in Size-exclusion/gel filtration chromatography

A

carbohydrate polymer

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

common carbohydrate polymer used in Size-exclusion/gel filtration chromatography

A

Dextran or agarose

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

The column matrix
constitutes about

A

65% of the column volume.
35 % Void volume

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

the remaining running buffer that fills
the column.

A

Void volume

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

1 void volume is
approximately
___fractions.

A

3

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

Separates proteins by their binding specificities or
on the basis of specific ligand interactions.

A

Affinity chromatography

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

Stationary phase in affinity chrom?

A

Polymeric material with bound ligands

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

those proteins without
affinity for the ligand flow through the column

A

Nonspecific proteins

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

The bound protein of particular interest is eluted
(washed out from the affinity column) by _______ or ______

A

adding a high concentration of ligand to the elution buffer or disrupting the binding interaction with changes in
pH.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

Separation is based on the molecular charge
(charge differences) of the protein molecule.

A

Ion-exchange chromatography

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

stationary phase in ion exchange chrom?

A

ion-exchanger

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Q

positively
charged anion-exchange
matrix

A

diethylaminoethyl (DEAE) cellulose

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
26
Q

negatively charged
cation-exchange matrix

A

carboxymethylcellulose
(CMC)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
27
Q

What is eluted first in ion exchanged using DEAE as matrix?

A

Anionic proteins

28
Q

What is eluted first in ion exchanged using CMC as matrix?

A

Anionic proteins

29
Q

a buffer that contains a high
concentration of an appropriate competing ion such as Na+ or Cl− that displaces the bound
protein.

A

elution buffer

30
Q

Enumerate in increasing order the efficiency of the protein purification techniques?

A

homogenization < Differential centrifugation< salting out with ammonium sulfate < size exclusion < ion-exchange < affinity chrom

31
Q

use of an electrical field to drive the
movement of some molecule (protein)
with a net charge rate based on their
charge mass-ratio and shape.

A

Electrophoresis

32
Q

approximate the molecular mass of a
protein and determine if the purified
protein contains more than 1
polypeptide chain.

A

Electrophoresis

33
Q

Types of electrophoresis

A
  1. Native polyacrylamide gel electrophoresis (PAGE)
  2. Isoelectric focusing
  3. SDS- polyacrylamide electrophoresis (SDS-PAGE)
  4. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE)
34
Q

Separation is based on their net negative charge
and their molecular mass and overall shape of
the protein

A

Native polyacrylamide gel electrophoresis (PAGE)

35
Q

Use of porous polyacrylamide

A

Native polyacrylamide gel electrophoresis (PAGE)

36
Q

Separation of proteins in native PAGE is visualized by staining with a dye such as

A

Coomassie Brilliant Blue G250

37
Q

a procedure used to separate proteins
on the basis of charge as a function of
pH (isoelectric point or pI)

A

Isoelectric focusing

38
Q

Use of __________ (mixture of organic acids and bases) to separate proteins in a gel

A

pH gradient

39
Q

Separation is
based on their
relative content
of positively and
negatively
charged residues

A

Isoelectric focusing

40
Q

Protein has a net ___________ when pH below its
isoelectric point.

A

positive charge

41
Q

Protein has a net ___________ when pH above its
isoelectric point.

A

negative charge

42
Q

Use of reducing agent and
sodium dodecyl sulfate (SDS)
which denature the protein –
secondary and tertiary
structures are disrupted.

A

SDS- polyacrylamide gel
electrophoresis (SDS-PAGE)

43
Q

SDS disrupts __________ interactions.

A

noncovalent

44
Q

1 molecule of SDS
associates with every
___ amino acid residues
( 1.4 g of SDS for every
gram of protein)

A

2

45
Q

dye used to
monitor the rate of
electrophoresis in SDS-PAGE

A

Bromophenol blue

46
Q

Separation is based on the
molecular mass of each
protein.

A

SDS-PAGE

47
Q

Estimate No. of Amino acids formula for SDS-PAGE

A

Estimate No. of Amino acids = Molecular mass ÷ 110

48
Q

Large proteins are best
separated by SDS-PAGE
using________
polyacrylamide gels,
whereas small proteins
resolve better in __________
polyacrylamide gels.

A

low-percentage, highpercentage

49
Q

By plotting the log molecular mass of known proteins versus migration distance in an SDS-PAGE system, the estimated mass of the unknown protein based
on its migration distance in the same
gel will be obtained. What is the x-axis and y-axis?

A

x = log molecular mass
y = migration distance

50
Q

Used to separate proteins on the basis of both
pI and molecular mass.

A

Two-dimensional polyacrylamide gel
electrophoresis (2-D PAGE)

51
Q

A protein mixture is separated in the first
dimension by ___________ and further
separated in second dimension by standard
___________

A

isoelectric focusing, SDS-PAGE.

52
Q

Identification of the amino-terminal
amino acid residue

A

Sanger method

53
Q

labels and removes only
the amino-terminal residue from a
peptide,leaving all other peptide bonds
Intact.

A

Edman
degradation

54
Q

use to separate and identify the free
amino acids formed after hydrolysis.

A

amino acid analyzer (HPLC)

55
Q

Cleavage of the disulfide bridges.

A

a. Oxidation of a cystine
b. Use of dithiothreitol (mercaptoethanol)
to reduce S-S bridges

56
Q

Identification of C-terminal amino acids

A

Use of carboxypeptidase enzymes

Carboxypeptidase A – C- terminal (except Pro)
Carboxypeptidase B – C- terminal for K and R

57
Q

cleaves only at the
carboxyl side of Met residues

A

Cyanogen bromide –

58
Q

cleaves N-side Asn and
Gly

A

Hydroxylamine HCl–

59
Q

cleaves phenol of Y, imidazole of H

A

Pauli’s reagent –

60
Q

Cleaves SH of Cys

A

Lead acetate

61
Q

N-side of K, guanidino of R

A

Sakaguchi Reagent

62
Q

C-side for any protein

A

Hydrazine

63
Q

proteolytic enzyme that cleaves only at carboxyl
side of Arg residues

A

Clostripain

64
Q

proteolytic enzyme that cleaves at N-side of Ile,
Leu, and Val

A

Thermolysin

65
Q

Determination of the sequence of
amino acids of the smaller peptides. Combining information from overlapping peptides to get
complete sequence.

A

Protein sequencing

Chem 193 Biochemistry C3 (4 decks)

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