Chapter 3 - Exploring proteins

Question 1

Proteome

Answer 1

Is the functional representation of the genome.

Signifies a more complex level of information content

Question 2

Peptide are used as

Answer 2

Hormones and pheromones
Neuropeptides
Antibiotics
Protection

Question 3

Separation of proteins relies on

Answer 3

Charge, size, affinity for a ligand, hydrophobicity, thermal stability.

Question 4

Differential configuration

Answer 4

Cells are disrupted in a homonizer and the homogenate is centrifuged. Used to purify protein from bacteria

Question 5

Dialysis

Answer 5

The protein is placed inside the dialysis bag, made up of cellulose, which is then submerged in a buffer solution. This does not distinguish between proteins. Often used to dilute salt from solution

Question 6

Gel filtration chromatography

Answer 6

Largest proteins come down first. Smaller proteins get into the porous beads made of agarose

Question 7

Ion-exchange chromatography

Answer 7

Purity is achieved based on distinct molecular properties. Based on their net charge. If a protein has a net positive charge at ph = 7 it will usually bind to a column of beads containing carboxylate groups

Question 8

Affinity chromatography

Answer 8

Can be used to isolate a protein. A protein mixture is passed through a column containing specific DNA sequences. Proteins with high affinity will bind. Then it’s washed with buffer to release unwanted proteins. Transcription factor is later released by adding a salt concentration solution

Question 9

Electrophoresis

Answer 9

A molecule with a net charge will move in an electric field. They are forced to move through the same matrix

Question 10

SDS page

Answer 10

Electrophoresis under denaturing conditions.
SDS is an an ionic detergent
B mercaptoethanol is added to reduce disulfide bonds

Question 11

Complex of SDS

Answer 11

With a denatured protein, it has a large net negative charge.
The loaded buffer is added and boiled
Migration through the gel is directly proportional to the logarithm of mass.

Question 12

Isoelectric focusing

Answer 12

Separated on the basis of their relative contents of acidic and basic residues. The isoelectric point of a protein is the pH at which its net charge is equal to zero

Question 13

Isoelectric focusing pt. Two

Answer 13

Positive charge when phpl

Question 14

How do you determine protein sequence?

Answer 14

Indirect: amino acid sequence
Direct: Edman degradation
Mass spectrometry

Question 15

Edman degradation

Answer 15

The N-terminal amino acid of a polypeptide is labeled.

Question 16

Tandem Mass Spect

Answer 16

Peptides can be fragmented by ions to generate a family of product ions. The carbonyl fragment of the cleaved peptide bond is ionized

Question 17

Proteins can be cleaved

Answer 17

Done by specific chemical reagents. They protein backbone at particular amino acid residues in a predictable manner

Question 18

Antibodies

Answer 18

Critical reagents for exploring the functions of proteins within the cell.

Question 19

Vitro protein synthesis

Answer 19

Built from C TO N terminance

Chloride reacts with C group

Question 20

How do you study protein structure?

Answer 20

X-ray chromatography

NMR

Question 21

An antibody

Answer 21

Is itself a protei, it is synthesized by vertebrates in response to the presence of a foreign substance called an antigen

Question 22

Immunoglobin

Answer 22

Consists of four chains, two heavy chains and red chains linked by disulfide bonds

Question 23

Polyclonal

Answer 23

Are heterogeneous mixtures of antibodies, each specific one of the various epitopes of an antigen

Question 24

Monoclonal

Answer 24

All identical, produced by clones of a single antibody-producing cell

Question 25

Preparation of monoclonal antibodies

Answer 25

Think of the rat. Hybridoma cells

Question 26

How to make polyclonal

Answer 26

Purify protein you want the antibody to grow in, inject protein in to animal, wait a few weeks while animals create antibodies for the antigen, collect blood

Question 27

Antibody techniques

Answer 27

ELISA
Western Blotting
Antibodies can be used as exquisitely specific analytic reagents to quantify the amount of a protein or other antigen present

Question 28

Indirect Elisa

Answer 28

Starts with an antigen coated well

Question 29

Sandwich Elisa

Answer 29

Starts with monoclonal antibody coated well

Question 30

Western blotting

Answer 30

To quantify very small quantities of proteins. The subject is subjected to electrophoresis on a SDS polyacrylamide gel. A primary and secondary antibody is added