Protein Techniques

Question 1

How do you determine the molecular weight of proteins?

Answer 1

Multiply the number of amino acids by 110

Question 2

What is the average MW of an amino acid?

Answer 2

110

Question 3

Ion exchange chromatography separates based on what?

Answer 3

Electric charge

Question 4

What determines charge in proteins?

Answer 4

pH

Question 5

Affinity chromatography separates proteins based on what?

Answer 5

Binding specificity

Question 6

How does affinity chromatography purify a specific protein?

Answer 6

By using a resin cross-linked with a specific ligand that tightly binds the protein, allowing all other proteins to be washed away

Question 7

Molecular sieve chromatography separates proteins based on what?

Answer 7

Size

Question 8

In molecular sieve chromatography, what size of molecules exits the column first? Why?

Answer 8

The larger molecules exit first because the smaller ones get stuck in the pores, and travel more slowly

Question 9

Gel electrophoresis separates proteins based on what?

Answer 9

Size

Question 10

How does gel electrophoresis work?

Answer 10

By using sodium dodecyl sulfate polyacrylamid gel, there is an overwhelming negative charge on the proteins. In an electrical field, proteins migrate toward the + pole (anode)

Question 11

Isoelectric focusing separates proteins based on what?

Answer 11

Isoelectric point

Question 12

In isoelectric focusing, proteins will migrate until they reach an area where their charge is what?

Answer 12

Zero - neutral

Question 13

Which test separates proteins based on both isoelectric point and molecular weight?

Answer 13

2D gel electrophoresis

Question 14

Which two tests are involved in 2D gel electrophoresis?

Answer 14

Isoelectric focusing

SDS-PAGE (gel electrophoresis)

Question 15

Proteomics is used to identify differences in protein profile of what?

Answer 15

Two different cell populations

Question 16

What does mass spectrometry measure?

Answer 16

Mass-to-charge ratio

Question 17

What are antibodies?

Answer 17

Proteins produced by B lymphocytes that destroy foreign antigens

Question 18

What are antigens?

Answer 18

Proteins (usually) that are recognized by other antibodies

Question 19

What size must a protein be in order to undergo sequencing?

Answer 19

Fewer than 50 amino acids

Question 20

Where does trypsin cleave?

Answer 20

On the carbonyl side of basic amino acids (Lys or Arg)

Question 21

Where does chymotrypsin cleave?

Answer 21

Carbonyl side of aromatic (Phe or Tyr)

Question 22

Where does S. aureus V8 protease cleave?

Answer 22

Carbonyl side of acidic amino acids (Glu or Asp)

Question 23

Where does cyanogen bromide cleave?

Answer 23

Carbonyl side of methionine

Question 24

In regards to protein sequencing, what is a fingerprint?

Answer 24

Regions of overlap resulting from protease fragmentation

Question 25

From which end are proteins sequenced?

Answer 25

N terminus

Question 26

From which end are proteins synthesized?

Answer 26

C terminus

Question 27

What are the three steps that are repeated in protein synthesis?

Answer 27

1. Protection/deprotection (NH2)
2. Activation (COOH)
3. Coupling (peptide bond)