Lecture 2: Tools of Cell Biology

Question 1

Most Biochemical analysis of cell function requires ___ amounts of material

Answer 1

large -can come from whole tissues and organs, or from cultured cells

Question 2

Most animals cells require a ____ to grow on and the appropriate mixture of ___.

Answer 2

surface, nutrients and growth factors

Question 3

Primary cultures

Answer 3

-Cells isolated directly from a particular tissue • Neurons, myobloasts, adrenal chromaffin cells etc – Usually do not survive many passages in culture – They age, and die in a process called replicative senescence

Question 4

Cell Lines

Answer 4

-Most commonly used cells lines have been modified to prevent death in culture – This is usually the result of alterations in normal cell cycle control mechanisms such as those that occur during cancer – These cell lines are referred to as “transformed cell lines” and grow in culture indefinitely and can be frozen and revived.

Question 5

Cell fractionation

Answer 5

• Cells can be separated into their component fractions • Cell extracts provide an accessible system to study cell functions

Question 6

____ can be used to fractionate cells

Answer 6

differential centrifugation

Question 7

What speeds do you spin to get which organelles?

Answer 7

10,000 g for 10 mins= Whole cell, nuclei, and cytoskeleton

20,000 g for 20 mins= mitochondria, lysosomes, peroxisomes

80-100,000 g for 1 hr= microsomes, small vesicles

150-200,000 g for 3 hrs= ribosomes, viruses, large macromolecules

Question 8

Density Sedimentation

Answer 8

a technique used to separate molecules on the basis of buoyant density.

Question 9

____ Chromatography Exploits Specific Binding Sites on Proteins

Answer 9

Affinity

Question 10

____ Provide an Easy Way to Purify Proteins

Answer 10

Genetically-Engineered Tags

Question 11

Purified ___ Systems are Required for the Precise Dissection of Molecular Functions

Answer 11

Cell-Free

Question 12

What are three types of chromatography? How do they work?

Answer 12

Question 13

What are the common epitote tags and how do they work?

Answer 13

• GST, MBP, His-tag, V5, Flag, Myc, HA

Question 14

Affinity purification approaches to study protein complexes

Answer 14

• Immunoprecipitation
• Glutathione-S-Transferase (GST) pull down

Question 15

What is GST?

Answer 15

Question 16

Sodium dodecyl sulfate (SDS)

Answer 16

a powerful ionic detergent

Question 17

Method of separation by SDS-PAGE

Answer 17

• Separation based on
charge to mass ratio
• Since the protein is
(usually) uniformly
coated with SDS,
separation is largely
based on mass (and
shape)

Question 18

How does isoelectric focusing work?

Answer 18

Question 19

What are two ways to analyze protein structure?

Answer 19

X-ray crystallography and NMR

Question 20

X-ray crystallography

Answer 20

x-ray diffraction pattern obtained from a protein crystal

Question 21

NMR spectroscopy

Answer 21

Nuclear Magnetic Resonance spectroscopy-The principle behind NMR is that many nuclei have spin and all nuclei are electrically charged. If an external magnetic field is applied, an energy transfer is possible between the base energy to a higher energy level (generally a single energy gap). The energy transfer takes place at a wavelength that corresponds to radio frequencies and when the spin returns to its base level, energy is emitted at the same frequency. The signal that matches this transfer is measured in many ways and processed in order to yield an NMR spectrum for the nucleus concerned.

Question 22

Northern Blot

Answer 22

RNA

Question 23

Southern blot

Answer 23

DNA

Question 24

Western Blot

Answer 24

proteins

Question 25

DNA is cut with…

Answer 25

restriction endonucleases

Question 26

Restriction digested DNA is annealed by….

Answer 26

Ligases using ATP, DNA polymerase fills in any gaps

Question 27

PCR

Answer 27

The polymerase chain reaction (PCR) is a technology in molecular biology used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.

-Denaturing with heat, cool to anneal primers, elongation

Question 28

Use of PCR to amplify a genomic or cDNA clone

Answer 28

Question 29

How to produce recombinant protein

Answer 29