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Biol200 > Purification, detection and characterization of protein > Flashcards

Purification, detection and characterization of protein Flashcards

1
Q

Protein purification and analysis manipulations depend on which physical and chemical properties? (4)

A

Mass or size (and shape)
Density
Electrical charge
Binding affinity

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2
Q

What are the separation methods used for protein purification and analysis? (3)

A

Centrifugation
Electrophoresis
Chromatography

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3
Q

What is centrifugation?

A

Centrifugation is a separation method that uses rapid spinning of the centrifuge tube to generate a centrifugal force that acts on particles suspended within the liquid medium of the tube.

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4
Q

What is the unit of measurement for centrifugal
force?

A

units of earth’s gravity (= 1 g)

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5
Q

What happens to particles that are denser than the suspending medium during centrifugation?

A

The g force will push the particles to the bottom of
the tube.

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6
Q

What happens to particles that are less dense than the suspending medium during centrifugation?

A

The g force will cause the particles to float toward
the top of the tube.

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7
Q

What happens to particles that have the same
density as the suspending medium during
centrifugation?

A

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

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8
Q

What are the two type of rotor used in centrifugation?

A

Swinging bucket rotor
Fixed-angle rotor

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9
Q

What is the purpose of centrifugation in protein
purification?

A

Centrifugation is used to separate particles based on their density, which can help to isolate the protein of interest from other cellular components.

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10
Q

The rate at which the supernatant is cleared of particles at a given centrifugal force depends on what?

A

the size/mass of the particle (for particles of similar shape)

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11
Q

What is the Svedburg unit?

A

It is a unit used to calculate the size of particles based on their sedimentation rate during centrifugation.

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12
Q

What is electrophoresis?

A

Electrophoresis is a separation method that uses an electric field to separate molecules based on their size and charge.

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13
Q

What is chromatography?

A

Chromatography is a separation method that uses a stationary phase and a mobile phase to separate molecules based on their interactions with the phases.

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14
Q

What is Equilibrium density gradient centrifugation?

A

It is a technique used to separate virus particles based on their density by creating a density gradient using high and low density sucrose solutions in a centrifuge tube.

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15
Q

At what density do virus particles band during Equilibrium density gradient centrifugation?

A

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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16
Q

During Electrophoresis, the direction of migration is determined by what?

A

the net charge

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17
Q

During Electrophoresis, the speed of migration is determined by what?

A

the net charge/mass ratio

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18
Q

What is SDS?

A

The anionic detergent sodium docecyl sulfate

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19
Q

What is the mechanism of action of SDS? (3)

A

-SDS denatures proteins by the interaction of its hydrophobic tail with hydrophobic amino acid side chains, disrupting the oil drop structure of proteins.
- The hydrophobic tail of SDS binds not only to hydrophobic residues, but also to itself, so it tends to coat the polypeptide chain in a uniform layer of SDS molecules.
-Because these are all negatively charged, the various parts of the SDS polypeptide chain repel each other and this further disrupts and unfolds the protein

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20
Q

What happens to multimeric proteins when they are
denatured by SDS?

A

All the chains of multimeric proteins are separated into individual denatured polypeptides.

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21
Q

What is SDS-PAGE?

A

SDS-PAGE stands for sodium dodecylsulfate polyacrylamide gel electrophoresis, a technique
used to separate proteins based on their size.

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22
Q

How does SDS-PAGE work?

A

SDS-PAGE denatures all proteins and uniformly binds them with negatively charged detergent, resulting in all proteins having about the same charge:mass ratio. The gel matrix impedes larger complexes, allowing for separation based on size.

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23
Q

What is the relationship between electrophoretic mobility and protein size in SDS polyacrylamide gel electrophoresis?

A

Migration rate is inversely related to protein size
(polypeptide length).

24
Q

How can post-translational modifications affect protein mobility during SDS polyacrylamide gel electrophoresis?

A

Phosphorylation of proteins by protein kinases can shift the mobility of the protein, and hence the apparent molecular weight.

25
Q

What is the isoelectric point of a protein?

A

The pH at which the sum of all charges equals zero.

26
Q

How does isoelectric focusing work?

A

A pH gradient is established using special buffers (ampholytes) immobilized in an acrylamide gel. Proteins subjected to electrical field migrate.

27
Q

What affects the isoelectric point of a protein?

A

amino acid composition

28
Q

What happens to acidic proteins and basic proteins
during isoelectric focusing?

A

Acidic proteins migrate towards the cathode, while basic proteins migrate towards the anode.

29
Q

What is the purpose of two-dimensional gel
electrophoresis?

A

To separate proteins based on both their isoelectric point and molecular weight, which allows for the simultaneous detection of many different proteins.

30
Q

What is the concept behind mass spectrometry? (3)

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 (m/z)

31
Q

Mass spectrometry allows high-precision determination of what?

A

the charge-to-mass ratio of ionized molecules

32
Q

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

A

electrospray ionization.

33
Q

The gas-phase ions generated by electrospray are separated in the ___________ into separate populations differing in _____.

A

mass analyzer
m/z

34
Q

What is proteomics?

A

Proteomics is the analysis of biological protein samples by mass spectrometry and bioinformatics in order to identify the population of proteins present in any given subcellular organelle.

35
Q

What is MS/MS (tandem MS)?

A

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

36
Q

What hapens during MS/MS (tandem MS)?

A

In MS-MS (tandem MS) peptide ions collected during a round
of MS are subjected to fragmentation, which occurs
preferentially at peptide bonds, and then subjected to a second
MS analysis.

37
Q

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

A

No, the fragmentation is partial and random,
meaning that on average only one (or a small
number of) peptide bond per molecule is broken.

38
Q

What is the “second dimension” of information
provided by the product ion spectrum in MS-MS?

A

The “second dimension” of information provided by
the product ion spectrum, analyzed computationally
with respect to known protein sequences, can
identify the amino acid sequence of the peptide ion.

39
Q

What is chromatography?

A

Chromatography is a method of separating components based on their differential interactions with an immobile (=solid) material, with a mobile phase (liquid or gas) moving continuously past the solid phase.

40
Q

What is the mobile phase used in protein chromatography?

A

The mobile phase used in protein chromatography
is generally liquid, usually an aqueous buffer.

41
Q

What is gel filtration chromatography?

A

Gel filtration is a separation method based on size, where the solid phase gel beads have pores of molecular dimensions, and the mobile phase freely
enters and exits the beads and flows between beads. Small proteins can enter and exit the beads through the pores, while large proteins cannot enter and run ahead of the small proteins.

42
Q

What is ion-exchange chromatography?

A

Protein molecules with a net electric charge will bind to immobile phase having the
opposite charge. Electrostatically-bound proteins can be released by flowing a salt solution through the column. The displacement of the protein by Na+ or Cl- is “ion exchange”.

43
Q

What is an antibody?

A

An antibody is a protein that recognizes, by highly-specific binding, a molecular target, called an epitope, present on the antigen molecule against which the antibody was raised.

44
Q

How many epitopes can a single antibody molecule
recognize?

A

A single antibody molecule recognizes a single
epitope.

45
Q

What is the practical limit to the number of different antibodies that can be raised and the number of different epitopes that can be recognized?

A

The number of possible different antibodies that can
be raised, and the number of different epitopes that
can be recognized, is practically infinite.

46
Q

What is antibody-affinity chromatography?

A

Antibodies specific for any particular protein can be covalently coupled to the solid phase. If a complex protein mixture is flowed through the column, only the protein to which the antibody binds will be retained. All the other proteins can be washed away and then the target protein can be released from the antibody by lowering the pH.

47
Q

What region of the primary antibody recognizes epitope of interest?

A

CDR
Complementarity Determining Region

48
Q

What region of primary antibody that is recognized by secondary antibody, from another species?

A

The constant region

49
Q

What are the steps for immunoblot?

A

-After a protein mixture has been electrophoresed through an SDS gel, the separated bands are transferred (blotted) from the gel onto a porous membrane.
-The membrane is flooded with a solution of an antibody (Ab1) specific for the protein of interest. The membrane is washed to remove unbound Ab.
-The membrane is incubald with a second antibody (Ab2) that specifically recognizes and binds to the first (Ab1).
-The location and amount of bound Ab2 are detected

50
Q

What is the mechanism of immunoprecipitation?

A

Recovery of protein complexes that bind to a specific antibody by precipitation, which contains the protein carrying the epitope recognized by the antibody and any partner proteins stably associated with that protein.

51
Q

What is co-immunoprecipitation?

A

It is a technique used to identify protein-protein interactions by isolating a protein of interest along with any partner proteins that are stably associated with it.

52
Q

What is indirect immunofluorescence?

A

It involves antibodies raised against the constant region of antibodies, which are chemically coupled (2ab) to a fluorochrome to permit detection in a fluorescence microscope.

53
Q

What is double-label immunofluorescence?

A

It is a technique that allows the simultaneous visualization of two proteins using two different
fluorescently labeled antibodies.

54
Q

What is GFP?

A

GFP is a single polypeptide chain that contains enzymatic activity that modifies some of its own amino acid side chains to generate the fluorochrome

55
Q

What is the use of GFP? (2)

A

-can be used as a reporter gene for transcriptional control elements
-can be made into fusion proteins to study intracellular protein localization.

56
Q

how to make the immunofluorescence experiment?

A

Inject purified mouse antibodies into a rabbit. The rabbit will recognize the mouse antibodies
as foreign and make antibodies against the mouse antibodies.
1-Prepare sample and place on microscope slide
2-incubate with primary Ab, wash away the unbound Ab
3-Incubate with fluorochrome-conjugated 2nd Ab. wash away unbound
4-Mount specimen and observe the fluorescence

57
Q

What are the steps on Co-IP experiment? (3) and what is the conclusion

A
  1. Non-SDS cell homogenate (natural, nondenatured proteins, bound to partners).
  2. Immunoprecipitate with anti GR antibody.
  3. Add SDS and do Western blot looking for presence of PPARa in immunoprecipitate (co-IP)

Conclusion: PPARalpha forms a stable complex with GR, but only when the latter is bound to its ligand.

Biol200 (12 decks)

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