Lecture 2: Tools of Cell Biology Flashcards Preview

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Flashcards in Lecture 2: Tools of Cell Biology Deck (29):
1

Most Biochemical analysis of cell function requires ___ amounts of material

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

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2

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

surface, nutrients and growth factors

3

Primary cultures

-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

4

Cell Lines

-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.

5

Cell fractionation

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

6

____ can be used to fractionate cells

differential centrifugation

7

What speeds do you spin to get which organelles?

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

8

Density Sedimentation

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

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9

____ Chromatography Exploits Specific Binding Sites on Proteins 

Affinity

10

____ Provide an Easy Way to Purify Proteins 

Genetically-Engineered Tags

11

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

Cell-Free

12

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

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13

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

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

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14

Affinity purification approaches to study protein complexes 

• Immunoprecipitation

• Glutathione-S-Transferase (GST) pull down 

15

What is GST?

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16

Sodium dodecyl sulfate (SDS) 

a powerful ionic detergent

17

Method of separation by SDS-PAGE

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

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18

How does isoelectric focusing work?

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19

What are two ways to analyze protein structure?

X-ray crystallography and NMR

20

X-ray crystallography

x-ray diffraction pattern obtained from a protein crystal

21

NMR spectroscopy

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.

22

Northern Blot

RNA

23

Southern blot

DNA

24

Western Blot

proteins

25

DNA is cut with...

restriction endonucleases

26

Restriction digested DNA is annealed by....

Ligases using ATP, DNA polymerase fills in any gaps

27

PCR

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

28

Use of PCR to amplify a genomic or cDNA clone 

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29

How to produce recombinant protein

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