Chapter 8: Analyzing Cells

Question 1

Method to study protein encoded in RNA

Answer 1

use bacteria (they’re simple)

Question 2

Method to study function of organelles/intracellular functions

Answer 2

use eukaryotic cells

don’t need something more complex, such as context in a tissue

Question 3

cells can be isolated from tissues

Answer 3

most realistic

1) disrupt extracellular matrix and cell-cell junctions (use proteolytic enzymes to digest proteins and chelator agents)
2) tease apart tissue into cells by gentle agitation

Question 4

methods to break open cells (don’t destroy the inside!)

Answer 4

high frequency sound, detergent (pokes holes in membrane), force cells through small tight spaces so they burst, glass vessel and pestle to shear open

Question 5

what do you end up with after breaking open cells?

Answer 5

a homogenate

Question 6

homogenate

Answer 6

mix of membrane components, cytoplasm, proteins

Question 7

cells can be grown in culture

Answer 7

aka in vitro (in glass)

can culture plant and animal cells

Question 8

purifying protein step one

Answer 8

extraction from inside the cell (break cells open)

Question 9

centrifugation

Answer 9

rotates cell extracts at high speeds

separates them by size and density

impure, multiple centrifugations can improve purity

Question 10

centrifugation where do larger objects go

Answer 10

large object = most centrifugal force = pellet at bottom

Question 11

low speed centrifugation

Answer 11

pellet contains whole cells, nuclei, cytoskeletons

Question 12

medium speed centrifugations

Answer 12

pellet contains mitochondria, lysosomes, peroxisomes

Question 13

high speed centrifugation

Answer 13

pellet contains microsomes, small vesicles

Question 14

very-high speed centrifugation

Answer 14

pellet contains ribosomes, viruses, large macromolecules

Question 15

supenatant

Answer 15

liquid above pellet

contains lighter molecules

Question 16

microsomes

Answer 16

resealed vesicles formed from fragments of rough and smooth ER

Question 17

column chromatography

Answer 17

separates by interaction w/ matrix

Question 18

ion exchange chromatography

Answer 18

separates on charge
resin is positive, positive flows through
can elute with diff concentrations of salt

Question 19

hydrophobic chromatography

Answer 19

hydrophobicity

column contains beads with hydrophobic chains, retard hydrophobic

Question 20

gel-filtration chromatography

Answer 20

size
large go through first, small get stuck in pores of beads
helps estimate size

Question 21

affinity chromatography

Answer 21

ability to bind to particular small molecules or to other macromolecules

EX: bead covered in polysaccharide, enzyme that operates on it will be retained

later can be washed off in pure form

EX: antibodies

Question 22

high-performance liquid chromatography (HPLC)

Answer 22

silica based resin
separates even at fast flow rates

great choice!

Question 23

protein purification by chromatography requires:

Answer 23

multiple steps in succession (usually)

a functional assay to follow purification so you know what fractions to keep