Lecture 3

Question 1

Quantification of cells expressing 2 different cell surface markers by FACS

Answer 1

• two different colours of fluorescence will be observed and recorded with each dot representing a single cell
• e..g. We are looking for the cells that express both proteins on their outermmebranes

Question 2

Cell cycle analysis by facs

Answer 2

• measuring the cells that have fluorescence using FACS
• blue fluorescence is indicative of the amount of DNA found in the cells

Question 3

Where are most cells found in cell cycle analysis by FAC

Answer 3

• in G1 which occurs before ell undergoes replication

Question 4

what stage willl have the highest intensity of hoechst stain fluorescence

Answer 4

• cells that have replicated their DNA but not fully divided

Question 5

what does the cell cycle analysis with FACS allow us to do

Answer 5

quantify cells at different stages of cell cycle

Question 6

how to disrupt plasma membrane

Answer 6

• mechanical homogenizatoin
• sonication
• pressure
• non ionic detergents e.g. triton x-100
• placing cells in hypotonic solution

Question 7

sonicaiton

Answer 7

ultrasound

Question 8

pressure

Answer 8

forcing cells through a small diameter tube to disrupt the plasma maebrane

Question 9

non ionic detegents

Answer 9

chemicals integrate into plasma mmebmrane causing its dissociation

Question 10

hypotonic solution

Answer 10

causes cell lyss

Question 11

bench top centriguation

Answer 11

• low speed

Question 12

ultracentrifuges

Answer 12

• spinning at high sepeds

Question 13

how is heat created in centriguation

Answer 13

when too spins around and around, takes stuff and forces it to bottom, spins so high interacts with oxygen molecules to create insnare heat

Question 14

why is it important to ensure balanced centriguation

Answer 14

• so that the weigh t is distributed and not become a projectile with the rotor breaking off its axis

Question 15

differential centrifugation

Answer 15

increasing centrifugal force (gravity) to isolate organelles based on their mass

Question 16

600g

Answer 16

seperates nuclei

Question 17

15 000 g

Answer 17

operates mitochondria, chloroplast, lysosome, peroxisomes

Question 18

100000g

Answer 18

PM, micrsomal fraction (fragments of ER) and large plyribsoomes

Question 19

300 000

Answer 19

ribosomal subunits, small polyribosomes, then can pour out soluble part of cytoplasm Icytosol)

Question 20

how does differential centriguation work

Answer 20

-successive series of centrifugal steps, increasing the force at each step startingf fro low and going higher, get progressively smaller stuff in the pellet

Question 21

how do isolate a specific organelle from the pellet

Answer 21

• use eq desnity gradient centirguation

Question 22

eq density gradient centriguation

Answer 22

• seperation based on ensity
• homogenate is applied to a gradient of sucrose
• apply a centreigual face
  -organelles will move down sucrose gradient and stop when they reach their equivalent density

Question 23

order of density for peroxisomes, mitochondria and lysosomes

Answer 23

peroxisomes (most dense), mito, lysoosmes (least dense)

Question 24

non ionic detergents

Answer 24

• disrupt the lipid bilayer r only

Question 25

ionic detergents

Answer 25

• dentature and disrupt ionic and hydrogen bonds

Question 26

detergents are ___ molecules

Answer 26

amphipathic

Question 27

amphipathic

Answer 27

both hydrophobic and hydroponics components

Question 28

which type of detergent should u use if u just want to disrupt the lipid bilayer

Answer 28

non ionic

Question 29

which detergent should u use if u want to denature and disrupt protein-protein interactions

Answer 29

ionic

Question 30

SDS page

Answer 30

• electoprhoertic separation of proteins

Question 31

SDS detergent

Answer 31

• Ionic detergent, immerse proteins with this, disrupts 3D structure and bind to Phobic portions within a protein and give a net negative charge to protein bc of negative sulphate anion

Question 32

charge to mass ratio

Answer 32

all of the proteins are forced into this negatively charged conformation

Question 33

how to see and quantity the proteins after SDS page

Answer 33

using a chemical stain.dye e,.g, coomassie brilliant blue or silver

Question 34

coomassie stain

Answer 34

• blue

Question 35

detecting a specific protein after SDS page

Answer 35

• antibody against the specific protein is added tot he gel
• transfer the proteins to the gel for better visualization/pentration
• incubate with the pimriary antibody which oldy bind to the proteins that are at a certain molecular weight
• use secondary antibody that is bound to horse radish peroxidase
• put inn dark box to capture light or x ray film to get an idea of the amount of a specific protein

Question 36

how does horse radish peroxidase help in the illumination of specific proteins

Answer 36

• it is bound to the secondary antibody, and reacts with substrate luminal to take it form non luminescent to luminescent