Chapter 2: 2.3 Isolation and Analysis of Cell Organelles and Molecules

Question 1

Describe:

FACS

Answer 1

Fluorescence Activated Cell Sorting
* Used to separate cells based on fluorescent labeling
* Cells can be labeled using fluorescently labeled probes or antibodies that bind to a cell surface protein, and then analyzed using FACS

Question 2

True or False:

In FACS, all the cells run through the maching will have the label

Answer 2

False, only some of those cells will have the label

Question 3

In FACS:

The cells pass through the detector…

Answer 3

One at a time

Question 4

In FACS:

What does the machine separates the cells by?

Answer 4

Separates the cells based on the levels of fluorescence detected

Question 5

In FACS:

What does it analyze?

Answer 5

Can be used to analyze the population of cells can be analyzed

Question 6

Describe:

Centrifugation

Answer 6

• Spin smaples at very high speeds
• Organelles separate based on density

Question 7

In centrifugation:

What does it do? What is the result?

Answer 7

Separates organelles separate based on density
* Heavier components go to the bottom and light components stay suspended

Question 8

In centrifugation:

What must be done first?

Answer 8

The cell needs to be lysed open first

Question 9

In centrifugation:

How does a cell need to be lysed to release a membrane bound protein?

Answer 9

Needs a detergent to break it open and release it

Question 10

Describe:

Differential centrifugation

Answer 10

Separate one layer at a time

Question 11

In differential centrifugation:

What does differential centrifugation take advantage of?

Answer 11

Takes advantage of the varying density differences between components

Question 12

What is SDS PAGE?

Answer 12

Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis
* A technique used to separate proteins

Question 13

In SDS PAGE:

What is SDS?

Answer 13

Sodium dodecylsulfate
* A detergent that coats the proteins and gives them a net negative charge

Question 14

List

The 5 steps in SDS PAGE

Answer 14

1. Protein samples are denatured to their primary state
2. Samples are treated with SDS to coat proteins in negative charges
3. Samples are loaded into an acrylamide gel (porous gel structure)
4. An electric current is applied where the negatively charged protein move towards to positive electric charge
5. Proteins move through the pores of the gel

Question 15

How do different proteins move through the pores of the gel in SDS PAGE?

Answer 15

• Smaller ones move faster
• Larger proteins move slower

Question 16

What is Western Blot?

Answer 16

• Used for detection of specific proteins

Question 17

In Western Blotting:

Describe the process

Answer 17

1. Transfer your SDS-PAGE onto a cellulose membrane
2. Add antibodies to the membrane that target specific proteins
3. Antibodies give off luminescence that can be photographed

Question 18

What type of membrane is normally used in Western blotting? What is used as an alternative?

Answer 18

Nitrocellulose membrane
* If proteins are hydrophobic, use PVDF membrane instead