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BIOL 200 > KH6 > Flashcards

KH6 Flashcards

1
Q

What are the physical and chemical properties of protein purification and analysis

A

Mass or size and shape
Density
Electrical charge
Binding affinity

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2
Q

What are separation methods in protein purification and analysis

A

Centrifgation
Electrophoresis
Chromatography

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3
Q

How is the centrifugal force that the centrifuge tube generates measured

A

In units of earth’s gravity (/g)

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4
Q

What does centrifugal force in centrifuges act on

A

Particles (down to molecular size) suspended within the liquid medium of the centrifuge tube

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5
Q

What kind of particle will be found at the bottom of the tube and why

A

Particles denser than the suspending medium will be pushed to the bottom by the g force

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6
Q

What kind of particles will be found at the top of the tube and why

A

If the particles are less dense than the suspending medium, the g force will float them

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7
Q

What happens when the particles are the same density as the suspending medium

A

They will not move in either direction but stay where they are

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8
Q

What is the rate at which the supernatant is cleared of particles at a given centrifugal force dependant on

A

The size/mass of the particles

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9
Q

What is the size unit calculated of a particle in a supernatant

A

Svedburg (S)

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10
Q

What is an examples of differential centrifugation

A

Separation of cellular contents by particle size/mass (mass of nucleus&raquo_space;> mitochondria)

Low speed centrifugation pellets nuclei, leaves mitos in supernatant

After: higher speed to recover mitos from supernatant

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11
Q

What is the process of purification of coronavirus

A

Two spins: first to take out mito at low speed, second at high speed to pellet virus particles

Resuspend pellet, apply equilibrium density gradient centrifugation

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12
Q

How does equilibrium density gradient centrifugation work

A
  1. Create a density gradient by smoothly mixing high and low density sucrose solutions while filling the centrifuge tube
  2. Apply the resuspended virus pellet to the top of the sucrose gradient
  3. Centrifuge at high speed: virus starts to move toward bottom of tube but stops when it hits a solution density equal to its own density
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13
Q

What is the direction of migration in a free solution in electrophoresis

A

Net charge

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14
Q

What is the speed of migration in a free solution in electrophoresis

A

Net charge/mass ratio

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15
Q

In what way can migration is gel electrophoresis be impeded by the gel

A

Larger molecules impeded more than small molecules

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16
Q

What does SDS do

A

Denatures proteins (unfolding) and coats them uniformly

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17
Q

How does SDS denature proteins

A

By the interaction of its hydrophobic tail with hydrophobic amino cid side chains disrupting the oil drop structure of proteins

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18
Q

What does the hydrophobic tail of SDS bind to and how does it coat the polypeptide chain in a uniform layer of SDS molecules

A

Hydrophobic residues and to itself (itself helps coat)

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19
Q

How do the various parts of the SDS polypeptide chain disrupt and unfold the protein

A

Everything is negatively charged and like charges repel

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20
Q

What are the characteristics of the unfolding and structural disruption caused by SDS

A

Completely denatures individual polypeptides, separates all the chains of multimeric protein into individual denatured polypeptides

21
Q

Does SDS has influence on denatured protein shape

A

No

22
Q

Why are all proteins negatively charged with about the same charge:mass ration

A

Because SDS is negatively charged and bunds uniformly to proteins

23
Q

How does a free solution compare to a polyacrylamide gel compare in electrophoretic mobility of particles and why

A

FS: all SDS protein complexes would have same electrophoretic mobility
PG: gel matrix impedes larger molecules more

24
Q

What is the relationship between migration rate in polyacrylamide gel and protein size (polypeptide length)

A

They are inversely proportional

25
Q

What is an example of a modification that can have an impact on protein mobility during SDS gel electrophoresis

A

Post-translational modifications (phosphorylation of proteins by protein kinases can in some cases shift the mobility of the protein and therefore molecular weight)

26
Q

What does the effect of phosphorylation of protein kinases on the mobility of protein result from

A

The phosphate group locally interfering with SDS binding

27
Q

What is the isoelectric point

A

pH at which the sum of all charges is 0

28
Q

What does isoelectric point depend on

A

Amino acid composition of each protein

29
Q

What kind of residues will give a high isoelectric point

A

Basic residues

30
Q

What kind of residues will yield a low isoelectric point

A

Acidic residues

31
Q

How is a pH gradient established in isoelectric focusing

A

Using special buffers immobilized in acrylamide gel

32
Q

When will proteins migrate in isoelectric focusing

A

When proteins are subjected to an electrical field

33
Q

How is a protein’s isoelectric point found using isoelectric focusing

A

Acidic protein that is positive migrates towards cathode (negative side) from a low pH side of strip OR Basic protein that is negative migrates towards anode (positive side) from a high pH side of strip

As the protein moves, the protein displays different charges in different regions of the strip until it is neutral, see what part of strip protein is on

Each line represents a protein at its isoelectric point

34
Q

What is two-dimensional gel electrophoresis

A

Isoelectric focusing followed by SDS PAGE

35
Q

Why after SDS PAGE in two dimensional gel electrophoresis are separated protein spots widely distributed and what does it reveal

A

In natural protein populations there is no relationship between isoelectric point and molecular weight

Reveals simultaneously many different proteins

36
Q

What are the two dimensions of two-dimensional gel electrophoresis and what do they separate by

A

Separate in first dimension by charge Separate in second dimension by size

37
Q

What kind of method is mass spectrometry

A

An analytical (not preparative) method

High precision determination of the charge to mass ratio of ionized molecules

38
Q

What are the 3 concepts of mass spectrometry

A
  1. Produce dispersed (individual molecules) ions in a gas phase
  2. Measure the acceleration of the ions in an electric or magnetic field
  3. Acceleration depends on the mass/charge ratio
39
Q

What does each amino acid and each oligopeptide have and what does it have if it carries a single charge

A

Characteristic molecular weight

single charge: molecular weight/charge = molecular weight

40
Q

What is a commonly used process for generating gas-phase ionized molecules

A

Electrospray ionization

41
Q

What happens to the gase-phase ions generated by electrospray

A

They are separated in the mass analyzer into separate populations different in molecular weight/charge (m/z)

42
Q

What is MS/MS

A

Recovering an ion, fragmenting it by high energy collision with an inert gas, and doing mass spectroscopy on the fragments

43
Q

What happens if peptide bonds are broken in amino acids

A

Peptide bonds are the weakest so id they are broken, amino acid side chains will still stay together

44
Q

What is the process of MS/MS

A
  1. peptide Ions collected during a round of MS
  2. Ions subjected to fragmentation occurring at peptide bonds
  3. ions subjected to a second MS analysis
45
Q

Does the fragmentation in MS/MS break every peptide bond in every molecule reducing the peptide to its individual amino acids

A

No, fragmentation is partial (on average only one peptide bond per molecule is broken) and random

46
Q

What does the “second dimension” of information provided by the product ion spectrum in MS/MS analyzed computationally with respect to known protein sequences identify

A

The amino acid sequence of the peptide ion

47
Q

What is proteomics

A

Analysis of biological protein samples by mass spectrometry and bioinformatics (computer analysis of DNA and protein sequences)

48
Q

Why is proteomics done

A

In order to identify the population of proteins present in any given sub cellular organelle

49
Q

Why are phosphorylated proteins heavier than non phosphorylated proteins

A

They are not necessarily too much heavier but they move a lot less as adding phosphates distrusts SDS from coating them in negative charge, making them less negative means they move less towards positive

BIOL 200 (13 decks)

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