Lecture 21 - Analyzing Cells, Molecules, and Systems

Question 1

Define cell culture.

Answer 1

-removal and isolation of cells from an organism and promoting subsequent growth with a favorable environment

Question 2

What is the process of sub-cellular fractionation?

Answer 2

• blending of tissue resulting in a suspension of different cell types
• centrifugation to separate cell types based on size and density
• lysis of cells
• ultracentrifugation to separate organelles

Question 3

How does centrifugation speed affect what is isolated and where it is?

Answer 3

Low centrifugation speed:

• largest/most dense components found in pellet
• smaller/less dense found in supernatent

High speed:

• smaller/less dense component located in pellet along with larger/more dense
• only smallest/least dense found in supernatent

Question 4

How does column chromatography work?

Answer 4

• mixture of substance are separated by flowing through a column
• separation occurs due to different interactions with the contents of the column which selectively slow flow of substances
• samples collected at bottom of column contain separated substances dissolved in column solvent

Question 5

Differentiate the two types of cell cultures.

Answer 5

Primary cell culture:

• derived directly the organism
• cell type must be isolated from other cell types present in the organ/tissue
• typically have a finite survival period

Established/continuous cell line:
-primary cell culture made immortal by transformation

Question 6

What are the different techniques to analyze proteins?

Answer 6

• SDS-PAGE
• Western blot
• ELISA
• mass spectrometry

Question 7

Differentiate the different types of column chromatography.

Answer 7

Ion-exchange:
-beads of a certain charge are in column and slow the rate of travel of oppositely charged substances

Gel-filtration:
-pourous beads are in the column and slow the rate of travel of small particles

Affinity:
-bead coated in substrate in column and only specific protein binds substrate

Question 8

What is SDS and what does it do?

Answer 8

Sodium dodecyl sulfate

• unfolds proteins
• gives proteins a uniform charge

Question 9

What does an SDS-PAGE do?

Answer 9

-separates proteins by size

Question 10

What is a Western blot?

Answer 10

• protein is isolated from a gel and then plated
• primary antibody is used to bind protein (antigen)
• secondary antibody is labeled and bind the primary antibody

Question 11

What is an ELISA?

Answer 11

• enzyme-linked immunosorbent assay

- tests for levels of specific antigen or antibody concentration using labeled antibodies

Question 12

What are the two different types of ELISA tests?

Answer 12

Indirect:
-well is antigen coated and binds specific antibody

Sandwich:
-well is antibody coated and binds specific antigen

Question 13

What is mass spectrometry?

Answer 13

• protein is broken into fragments (Tryptic digestion)

- each fragment is detected individually