Module 04: Cellular Analysis

Question 0

What is done in mechanical lysing?

Answer 0

Rotating blades grind and disperse cells and tissues

Question 1

___________ stop proteins and lipids from sticking together (solubilization).

Answer 1

Detergents

Question 2

Once the cell is lysed and mixed into a homogenous solution, it is called:

Answer 2

Cell lysate (or cell homogenate)

Question 3

centrifugation (n.)

Answer 3

the separation of particles depending on size or density

Question 4

What is the unit of the sedimentation coefficient?

Answer 4

Svedberg units (S)

Question 5

What does sedimentation coefficient measure?

Answer 5

The rate of particle sedimentation

Question 6

Increasing sedimentation coefficient ________ the rate of sedimentation.

Answer 6

increases

Question 7

What is relative centrifugal force (RCF) measured in?

Answer 7

xg, the relative multiple to the force of gravity

Question 8

What is the equation for RCF?

Answer 8

RCF = 1.12 x R x (rpm/1000)²

Question 9

What effect does doubling the radius have on RCF? (AEC)

Answer 9

Double the RCF

Question 10

microsome (n.)

Answer 10

a vehicle derived from endomembrane system

Question 11

polysome (n.)

Answer 11

a single mRNA with multiple ribosomes attaches, each synthesizing a polypeptide from the same mRNA

Question 12

differential centrifugation (n.)

Answer 12

sequential centrifugation at higher so for longer times

Question 13

Give a differential centrifugation sequence.

Answer 13

1000 xg for 10min; 20,000 xg from 20min; 80,000 xg for 1h; 200,000 xg for 2h

Question 14

cellular homogenate (n.)

Answer 14

suspension of all material from broken cells

Question 15

What do you get after the first round of DC?

Answer 15

Postnuclear supernant, and pellet containing nuclei and unbroken cells

Question 16

density gradient centrifugation (n.)

Answer 16

centrifuging using a concentration gradient (e.g., sucrose)

Question 17

Density of 2.3 M sucrose

Answer 17

1.3 g/cm³

Question 18

Density of lysosomes

Answer 18

1.1 g/cm³

Question 19

Density of mitochondria

Answer 19

1.2 g/cm³

Question 20

Density of peroxisomes

Answer 20

1.3 g/cm³

Question 21

What do you get after the second round of DC?

Answer 21

Postmitochondrial supernant, and pellet containing large particles (mitochondria, lysosomes, and peroxisomes)

Question 22

What do you get after the third round of DC?

Answer 22

Postmicrosomal supernant, and pellet containing fragments of cell membranes, smooth ER, and rough ER

Question 23

What do you get after the fourth round of DC?

Answer 23

Cytosol, and pellet containing viruses, free ribsosomes, and large macromolecules

Question 24

What does coomassie stain?

Answer 24

Protein

Question 25

What color does coomassie stain?

Answer 25

Brilliant blue

Question 26

What does SDS do?

Answer 26

Denatures proteins

Question 27

native state (n.)

Answer 27

folded and functional state of protein

Question 28

What does DTT stand for?

Answer 28

Dithiothreitol

Question 29

How many polypeptide chains comprise insulin?

Answer 29

Two polypeptide chains

Question 30

What are the separate polypeptide chains insulin held together by?

Answer 30

Disulfide bonds

Question 31

What kind of agent is DTT?

Answer 31

A reducing agent

Question 32

What are the steps of a Western blot/immunoblot?

Answer 32

SDS polyacrylamide gel electrophoresis, protein blot on nitrocellulose, antibody labeling, antibody detection