Lec 5

Question 1

Unicellular eukaryotes

example

Answer 1

S cerevisiae

Yeast

Cultures are grown in YEPD liquid media or on YEPD agar plates

Question 2

YEPD media

Answer 2

Yeast Extract

Peptone (milk proteins)

Dextrose (glucose)

Question 3

Multicellular organisms

Answer 3

Most can grow in vitro if they have enough to survive

Question 4

Media (food) to grow multicellular organisms includes

Answer 4

Amino acids

Vitamins

Salts

Glucose (carbon source/ATP generation)

Antibiotics (kill bacteria)

Phenol red for pH

Growth factor proteins (allows cells to reproduce)

Question 5

Subcellular fractionation

Purpose

Answer 5

The isolation and purification of cellular organelles and macromolecules using a centrifuge

Question 6

Subcellular fractionation

steps

Answer 6

Homogenization

Isolation

Purification

Question 7

Centrifuge

Answer 7

Can spin up to 15k rpm

Question 8

Subcellular fractionation needs

Answer 8

An ultra centrifuge

Question 9

Centrifuge needs to spin in a

Answer 9

Vacuum to prevent friction as well as armor plating because if something goes wrong and the rotor is released, it will come off at a very high speed. A refrigerator is also needed to offset heat of friction

Question 10

Homogenization

Answer 10

Breaks plasma membrane to release the cell contents

Ends with cell homogenate

Question 11

4 methods for homogenization

Answer 11

High frequency sound to vibrate them into pieces

Mild detergent to disrupt plasma membrane

Constriction to force cells through and break the ones that are too big to fit

Mechanical shearing

High frequency is the most common

Question 12

Isolation involves

Answer 12

Differential centrifugation

Separates cell components according to whether they sediment or not

Involves spinning the cell components

Heavy things end up at the bottom of the tube while others will stay floating around

Heavier components that fall are known as pellets

Might need to spin more than once to obtain pellets which is what we are looking for

Question 13

Microsome

Answer 13

fragments of ER

Question 14

Purification involves

Answer 14

Velocity centrifugation

Everything tried to move to bottom of tube

Larger/heavier things move faster

Once it is done, a hole is poked into the bottom and what is at the bottom is collected into different tubes

Question 15

How do you know what tub has the organelle you are looking for

Answer 15

Experience

Optical density

Microscopic examination

Question 16

Another method of purification

Answer 16

Equilibrium centrifugation

Things are spun again but they move according to buoyancy rather than mass

A gradient of different concentration batches of sucrose is made in order to prepare this

Heavier stuff ends up at bottom lighter is on top

Final steps are the same as velocity of centrifugation

Question 17

Total possible method pathways of subcellular fractionation

Answer 17

8