Exam Two: Ch 5 Learning Objectives

Question 1

Define salting out

Answer 1

protein precipitates as a result of increase in the salt and a decreased solubility in the solvent

Question 2

Solubility is dependent on?

Answer 2

dissolved salts
polarity of solvent
pH
temperature

Question 3

Match the protein characteristic with the purification procedure
a. solubility
b. ionic charge
c. polarity
d. size
e. binding specificity
f. salting out
g. ionic exchange chromatography
h. electrophoresis
i. hydrophobic interaction chromatography
j. SDS-PAGE
k. Gel-Filtration chromatography
l. affinity chromatography

Answer 3

a - f
b - g,h,
c - i
d - j,k
e - l

Question 4

Define the mobile and stationary phase in chromatography

Answer 4

liquid - mobile phase
stationary - porous solid matrix

Question 5

How does chromatography work?

Answer 5

based on target protein interaction with stationary phase

Question 6

What are the two types of ion exchange?

Answer 6

anion
cation

Question 7

Define an anion exchanger.

Answer 7

anions bind to cationic group using DEAE, most negative charge is eluted last

Question 8

Define a cationic exchanger

Answer 8

cations bind to anionic groups using CM - most positively charged ion is eluted first

Question 9

How does gel filtration chromatography work?

Answer 9

size exclusion - larger molecules elute first

Question 10

What is the stationary phase of gel filtration?

Answer 10

gel beads with narrow pores

Question 11

What is the stationary phase of gel filtration made out of?

Answer 11

agarose or polyacrylamid

Question 12

Describe affinity chromatography.

Answer 12

desired proteins bind to immobilized ligands and others are washed out

Question 13

Describe hydrophobic interaction chromatography

Answer 13

separates proteins primarly based on presence of non-polar residues on the surface

Question 14

What is hydrophobic interaction chromatography used for?

Answer 14

purify either a protein (assoc w fatty acids) or nonpolar molecules with salt (nonpolar group interacts and is excluded)

Question 15

Describe SDS PAGE electrophoresis

Answer 15

Sodium dodecyl sulfate (SDS) denatures proteins by disrupting non-covalent interactions in a polypeptide

Question 16

SDS PAGE is __ SDS per __ amino acid residues

Answer 16

1;2

Question 17

How does SDS PAGE separate ?

Answer 17

according to MW

Question 18

What is the function of Mercaptoethanol?

Answer 18

used to cleave disulfide bonds in an SDS PAGE electrophoresis

Question 19

What are two endopeptidases?

Answer 19

trypsin and chymotrypsin

Question 20

Differentiate between an endopeptidase and an exopeptidase

Answer 20

endo - catalyze hydrolysis of INTERNAL peptide bonds
exo - catalyze hydrolysis of N or C terminal residues

Question 21

Where does Trypsin cleave?

Answer 21

C side of positively charged residues
Arg and Lys if next is NOT Pro

Question 22

Where does Chymotrypsin cleave?

Answer 22

C side of Phe, Trp, and Try if next residue is NOT Pro

Question 23

What is Cyanogen Bromide (CNBR)? And what is it used for?

Answer 23

chemical reagent used to cleave C side of Met residues

Question 24

What is the basis of an ELISA procedure?

Answer 24

a target protein in a complex mixture is detected by binding of antibodies based on reactions between proteins and antibodies

Question 25

What are the steps of an ELISA procedure?

Answer 25

1. Incubate first antibody with protein containing sample 2. Add a second antibody that is covalently linked to an assayble enzyme 3. Wash and assay enzyme activity

Question 26

For an ELISA procedure, the amount of substrate converted is equal to?

Answer 26

the amount of protein present

Question 27

What is the purpose of Protein sequencing?

Answer 27

separates subunits by cleaving polypeptide chains

Question 28

How does Edman Degradation work?

Answer 28

removes an N-terminus amino acid residues one at a time

Question 29

What does Edman Degradation reveal?

Answer 29

number of different types of subunits

Question 30

Describe the process of Edman Degradation.

Answer 30

PITC reacts with N-terminal group under mildly alkaline conditions yields PTC adduct - converts to PTH determine amino acid by chromatography

Question 31

Describe the four steps used to determine the amino acid sequence

Answer 31

1. separate and identify individual amino acids by HPLC 2. identify N-terminal and C-terminal amino acids 3. cleave at specific sides 4. cleave at different specific sites 5. use 3 and 4 to determine the sequence