LAB 1

Question 1

What is the basic unit of life that contains various organelles?

Answer 1

Cells

Organelles include mitochondria, Golgi apparatus, endoplasmic reticulum, and chloroplast.

Question 2

What technique is used to isolate organelles in a purified state?

Answer 2

Differential centrifugation

This technique relies on the density and size of organelles.

Question 3

What happens to denser organelles in a centrifugal field?

Answer 3

They travel to the bottom of the centrifuge tube

This is due to their density compared to the surrounding medium.

Question 4

The general procedure for isolation of organelles starts with

Answer 4

Homogenization / cell disruption

Question 5

What is the purpose of using an isotonic solution during cell disruption?

Answer 5

Prevents the rupture of membrane vesicles due to osmosis

Common isotonic solution used is sucrose solution.

Question 6

Mechanical disruption can be done by using

Answer 6

Homogenizer or mortar and pestle

Question 7

What should be the first step in the isolation of organelles?

Answer 7

Cell disruption or homogenization

This can be done using a homogenizer or mortar and pestle.

Question 8

What is the function of low centrifugal force in the initial centrifugation?

Answer 8

Separates large cellular components like nuclei and cellular debris

This step ensures smaller organelles remain in the supernatant.

Question 9

What are the cytoplasmic organelles collected in the next centrifugation step?

Answer 9

Mitochondria, chloroplasts, peroxisomes, lysosomes

These are isolated at higher centrifugal forces.

Question 10

What is ultracentrifugation used for?

Answer 10

Isolating smaller cellular components like microsomes and ribosomes

Requires around 75000 rpm of speed.

Question 11

How can the composition of cellular fractions be analyzed?

Answer 11

By microscopic examination or measuring specific proteins

Each organelle has specific proteins that can be quantitatively measured.

Question 12

Organelles isolated through Differential centrifugation retain their normal activity as long as

Answer 12

They are not exposed to denaturing conditions

Question 13

The microsomes and ribosomes can be isolated by ultracentrifugation at what speed

Answer 13

75000 pm of speed

Question 14

What is the role of methylene blue in the assay?

Answer 14

Detects the presence of chloroplasts in the fractions

Methylene blue binds to negatively charged parts of cells.

Question 15

Why is it important to perform the experiment on ice?

Answer 15

Maintains chloroplast integrity and minimizes oxidative stress

Low temperatures prevent membrane destabilization.

Question 16

What are the four purposes of using 0.5 M sucrose in the homogenization step?

Answer 16

Creates an isotonic environment
Stabilizes chloroplast structure
Aids in organelle separation
Protects chloroplast enzymes

Each purpose contributes to the successful isolation of chloroplasts.

Question 17

What happens during the first low-speed centrifugation?

Answer 17

Large debris like cell walls and nuclei are removed

Smaller organelles remain in the supernatant for further centrifugation.

Question 18

How does centrifugal force affect organelle separation?

Answer 18

Greater centrifugal force causes heavier objects to precipitate more readily

This allows for effective separation of organelles based on density.

Question 19

What is the expected composition of the supernatant after medium-speed centrifugation?

Answer 19

Contains cytosolic components while chloroplasts are in the pellet

Chloroplasts are denser than most organelles but lighter than nuclei.

Question 20

What is the isoelectric point (IpH) of a protein?

Answer 20

The pH at which a protein has a net charge of zero

At this pH, proteins are least soluble.

Question 21

What is the salting-out method in protein isolation?

Answer 21

Precipitating proteins by saturating the solution with ammonium sulfate

This method utilizes the ionic strength of the solution.

Question 22

What is the primary mechanism of the isoelectric precipitation method?

Answer 22

Variation in pH changes the ionization state of functional groups in a protein

This affects protein solubility.

Question 23

What should be done with the centrifuge tubes before centrifugation?

Answer 23

They should be balanced

This ensures even weight distribution and prevents damage to the centrifuge.

Question 24

What is the basic principle of centrifugation?

Answer 24

Separation of molecules having different densities by spinning them at high speed

This creates a centrifugal force that causes sedimentation.

Question 25

What defines the relative centrifugal force (RCF)?

Answer 25

A common measurement of centrifugal force in terms of Earth's gravitational force ## Footnote RCF is reported as a multiple of g.

Question 26

What is the function of high-speed centrifuges?

Answer 26

To achieve higher speeds for the sedimentation of particles ## Footnote Speeds range from 1000-25000 rpm.

Question 27

What happens to proteins during the salting-out process?

Answer 27

They aggregate and precipitate out of the solution ## Footnote Increased ionic strength reduces protein-water interactions.

Question 28

What is the purpose of grinding leaves in the chloroplast isolation process?

Answer 28

To disrupt the cells and release chloroplasts ## Footnote This mechanical disruption is essential for effective homogenization.

Question 29

What is the expected outcome of the methylene blue decolorization assay?

Answer 29

To measure the time it takes for chloroplasts to decolorize the solution ## Footnote Quick decolorization indicates high chloroplast activity.

Question 30

What should be done if the suspension decolorizes the methylene blue within seconds?

Answer 30

Dilute it with the isolation medium ## Footnote This ensures accurate measurement of chloroplast activity.

Question 31

What is the role of enzymes in the chemical and enzymatic methods of cell disruption?

Answer 31

To selectively release molecules by disrupting cell membranes ## Footnote Enzymes can effectively break down specific cellular components.

Question 32

What are the characteristics of eukaryotic cells?

Answer 32

- Membranous organelles - Specific functions - Vacuoles in plants ## Footnote Eukaryotic cells have complex structures compared to prokaryotic cells.

Question 33

What is present as organelles

Answer 33

- Mitochondria - Golgi Apparatus - ER - Lysosomes - Chloroplast (plants) - Vacuoles (Plants)

Question 34

What is the significance of the Nobel Prize awarded in 1974 in relation to cell biology?

Answer 34

Recognized discoveries concerning the structural and functional organization of the cell ## Footnote Awarded to Albert Claude, Christian de Duve, and George E. Palade.

Question 35

Who won the Nobel prize in physiology or medicine in 1974

Answer 35

Albert Claude, Christian de Duve, George E. Palade AC, CD, GP aircon, cd, gopro AAHAHHA

Question 36

What is the effect of temperature during centrifugation?

Answer 36

Maintains the integrity of cellular components ## Footnote High temperatures can denature proteins and disrupt organelles.

Question 37

Disrupting cells and tissues by applying a force not inherent to the sample is considered a

Answer 37

Mechanical disruption method

Question 38

Grinding uses

Answer 38

Mortar and Pestle

Question 39

Shearing use

Answer 39

Homogenizer

Question 40

Beating

Answer 40

Bead Beater

Question 41

Shocking

Answer 41

Sonicator

Question 42

Chemicals, such as surfactants, detergents, and chaotropic agent, can

Answer 42

- thoroughly disrupt cells - make the cell membrane permeable - or under appropriate conditions enter the cell to selectively release molecules

Question 43

Can also be used to disrupt cells

Answer 43

Enzyme

Question 44

SDS meaning

Answer 44

Sodium dodecylsulfate

Question 45

CTAB means

Answer 45

cetyltrimethylamminumbromide

Question 46

What is the speed of high speed centrifuges

Answer 46

(1000-25000rpm, ~50000 xg)

Question 47

High speed centrifuges types

Answer 47

fixed angle, swinging bucket, vertical rotor

Question 48

Ultracentrifuges speed

Answer 48

20000-80000 rpm (600000 x g)

Question 49

Ultracentrifuges uses

Answer 49

Spin chamber is refrigerated and placed under a high vacuum

Question 50

Low speed centrifuges are used for

Answer 50

sedimentation or relatively heavy particles

Question 51

Low speed centrifuges speed ranges from

Answer 51

4000 – 5000 rpm (-3000 x g)

Question 52

What centrifuges operates at room temperature

Answer 52

Low speed centrifuge

Question 53

Low speed centrifuge

Answer 53

Swinging bucket or fixed angle

Question 54

Chemical structure of a strong ionic detergent, such as sodium dodecyl sulfate, showing the

Answer 54

hydrophobic and hydrophilic sections and micelle formation

Question 55

SDS exists as individual monomers

Answer 55

below critical micelle concentrations (CMC) while assembling as micelles above the CMC (adapted from life canvas technologies)

Question 56

Basic principle of centrifugation

Answer 56

A particle (cells, organelles, macromolecules) is subjected to high centrifugal force when it is rotated at high rate of speed

Question 56

Centrifugal force equation

Answer 56

F=𝒎𝝎2𝒓

Question 57

F = mw2r define

Answer 57

F= Intensity of centrifugal force M= effective mass of sedimenting particle W= angular velocity of rotation r = distance of the migrating particle from the central axis of rotation

Question 58

A common measurement of F, in terms of the earth’s gravitational force

Answer 58

Relative Centrifugal force, RCF

Question 59

The relationship between RCF and revolutions per minute (rpm) is defined by the equation below:

Answer 59

RCF = (rpm^2) (1.119x10^-5) (r) r = radius OK DONT FORGET

Question 60

RCF unit

Answer 60

x g