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Biochemistry > Exam Two: Ch 5 Learning Objectives > Flashcards

Exam Two: Ch 5 Learning Objectives Flashcards

1
Q

Define salting out

A

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

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2
Q

Solubility is dependent on?

A

dissolved salts
polarity of solvent
pH
temperature

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3
Q

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

A

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

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4
Q

Define the mobile and stationary phase in chromatography

A

liquid - mobile phase
stationary - porous solid matrix

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5
Q

How does chromatography work?

A

based on target protein interaction with stationary phase

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6
Q

What are the two types of ion exchange?

A

anion
cation

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7
Q

Define an anion exchanger.

A

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

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8
Q

Define a cationic exchanger

A

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

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9
Q

How does gel filtration chromatography work?

A

size exclusion - larger molecules elute first

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10
Q

What is the stationary phase of gel filtration?

A

gel beads with narrow pores

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11
Q

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

A

agarose or polyacrylamid

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12
Q

Describe affinity chromatography.

A

desired proteins bind to immobilized ligands and others are washed out

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13
Q

Describe hydrophobic interaction chromatography

A

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

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14
Q

What is hydrophobic interaction chromatography used for?

A

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

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15
Q

Describe SDS PAGE electrophoresis

A

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

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16
Q

SDS PAGE is __ SDS per __ amino acid residues

A

1;2

17
Q

How does SDS PAGE separate ?

A

according to MW

18
Q

What is the function of Mercaptoethanol?

A

used to cleave disulfide bonds in an SDS PAGE electrophoresis

19
Q

What are two endopeptidases?

A

trypsin and chymotrypsin

20
Q

Differentiate between an endopeptidase and an exopeptidase

A

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

21
Q

Where does Trypsin cleave?

A

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

22
Q

Where does Chymotrypsin cleave?

A

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

23
Q

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

A

chemical reagent used to cleave C side of Met residues

24
Q

What is the basis of an ELISA procedure?

A

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

25
Q

What are the steps of an ELISA procedure?

A
  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
26
Q

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

A

the amount of protein present

27
Q

What is the purpose of Protein sequencing?

A

separates subunits by cleaving polypeptide chains

28
Q

How does Edman Degradation work?

A

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

29
Q

What does Edman Degradation reveal?

A

number of different types of subunits

30
Q

Describe the process of Edman Degradation.

A

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

31
Q

Describe the four steps used to determine the amino acid sequence

A
  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

Biochemistry (8 decks)

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