PROTEIN SEPARATION AND PURIFICATION

Question 1

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

Answer 1

Homogenization of
tissue or microbial
cells to produce cell
extracts

Question 2

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

Answer 2

Differential Centrifugation

Question 3

how many subcellular fractions are obtain in differential centrifugation

Answer 3

4

Question 4

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

Answer 4

Specific gravity

Question 5

Formula of specific gravity

Answer 5

total unit of activity / total protein

Question 6

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

Answer 6

Fractionation by Salting Outs

Question 7

salt used in fractionation

Answer 7

ammonium sulfate

Question 8

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

Answer 8

Dialysis

Question 9

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

Answer 9

Separation and Purification by Column
Chromatography

Question 10

Types of Column
Chromatography

Answer 10

1. Size-exclusion chromatography/gel
   filtration chromatography
2. Affinity Chromatography
3. Ion-exchange chromatography

Question 11

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

Answer 11

Column
Chromatography

Question 12

Separates proteins according to
size.

Answer 12

Size-exclusion/gel filtration
chromatography

Question 13

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

Answer 13

Larger proteins

Question 14

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

Answer 14

carbohydrate polymer

Question 15

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

Answer 15

Dextran or agarose

Question 16

The column matrix
constitutes about

Answer 16

65% of the column volume.
35 % Void volume

Question 17

the remaining running buffer that fills
the column.

Answer 17

Void volume

Question 18

1 void volume is
approximately
___fractions.

Answer 18

3

Question 19

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

Answer 19

Affinity chromatography

Question 20

Stationary phase in affinity chrom?

Answer 20

Polymeric material with bound ligands

Question 21

those proteins without
affinity for the ligand flow through the column

Answer 21

Nonspecific proteins

Question 22

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

Answer 22

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

Question 23

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

Answer 23

Ion-exchange chromatography

Question 24

stationary phase in ion exchange chrom?

Answer 24

ion-exchanger

Question 25

positively charged anion-exchange matrix

Answer 25

diethylaminoethyl (DEAE) cellulose

Question 26

negatively charged cation-exchange matrix

Answer 26

carboxymethylcellulose (CMC)

Question 27

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

Answer 27

Anionic proteins

Question 28

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

Answer 28

Anionic proteins

Question 29

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

Answer 29

elution buffer

Question 30

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

Answer 30

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

Question 31

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.

Answer 31

Electrophoresis

Question 32

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

Answer 32

Electrophoresis

Question 33

Types of electrophoresis

Answer 33

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

Question 34

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

Answer 34

Native polyacrylamide gel electrophoresis (PAGE)

Question 35

Use of porous polyacrylamide

Answer 35

Native polyacrylamide gel electrophoresis (PAGE)

Question 36

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

Answer 36

Coomassie Brilliant Blue G250

Question 37

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

Answer 37

Isoelectric focusing

Question 38

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

Answer 38

pH gradient

Question 39

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

Answer 39

Isoelectric focusing

Question 40

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

Answer 40

positive charge

Question 41

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

Answer 41

negative charge

Question 42

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

Answer 42

SDS- polyacrylamide gel electrophoresis (SDS-PAGE)

Question 43

SDS disrupts __________ interactions.

Answer 43

noncovalent

Question 44

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

Answer 44

2

Question 45

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

Answer 45

Bromophenol blue

Question 46

Separation is based on the molecular mass of each protein.

Answer 46

SDS-PAGE

Question 47

Estimate No. of Amino acids formula for SDS-PAGE

Answer 47

Estimate No. of Amino acids = Molecular mass ÷ 110

Question 48

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

Answer 48

low-percentage, highpercentage

Question 49

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?

Answer 49

x = log molecular mass y = migration distance

Question 50

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

Answer 50

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

Question 51

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

Answer 51

isoelectric focusing, SDS-PAGE.

Question 52

Identification of the amino-terminal amino acid residue

Answer 52

Sanger method

Question 53

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

Answer 53

Edman degradation

Question 54

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

Answer 54

amino acid analyzer (HPLC)

Question 55

Cleavage of the disulfide bridges.

Answer 55

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

Question 56

Identification of C-terminal amino acids

Answer 56

Use of carboxypeptidase enzymes Carboxypeptidase A – C- terminal (except Pro) Carboxypeptidase B – C- terminal for K and R

Question 57

cleaves only at the carboxyl side of Met residues

Answer 57

Cyanogen bromide –

Question 58

cleaves N-side Asn and Gly

Answer 58

Hydroxylamine HCl–

Question 59

cleaves phenol of Y, imidazole of H

Answer 59

Pauli’s reagent –

Question 60

Cleaves SH of Cys

Answer 60

Lead acetate

Question 61

N-side of K, guanidino of R

Answer 61

Sakaguchi Reagent

Question 62

C-side for any protein

Answer 62

Hydrazine

Question 63

proteolytic enzyme that cleaves only at carboxyl side of Arg residues

Answer 63

Clostripain

Question 64

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

Answer 64

Thermolysin

Question 65

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

Answer 65

Protein sequencing