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Molecular Cell Biology > Molecular cell biology uwindsor lecture 3 > Flashcards

Molecular cell biology uwindsor lecture 3 Flashcards

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1
Q

What are some of the advantages of growing cells in vitro?

A

Individual cells grown in culture with specific media can simulate actual phenotype in vivo. Advantages include purity of culture as well.

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2
Q

What are some of the disadvantages of growing cells in vivo?

A

Cells don’t interact with all neighbours the same as in vivo.

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3
Q

What is the point of purifying proteins?

A

Can study them in vitro, best method of studying a biological process by determining all the components of said process. If proteins are isolated, we can understand the system.

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4
Q

What is an in vitro assay?

A

Reconstitute a specific cellular event in a test tube.

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5
Q

How does one accomplish an in vitro assay?

A

Start with crude cell extracts, add ATP and use a method to assay for the specific protein activity.

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6
Q

How does one assay for DNA replication?

A

Add radioactive nucleotides, see if it goes from acid soluble to insoluble fraction (indicating the incorporation into the DNA)

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7
Q

How does one separate cell into their component fractions?

A

Centrifugation.

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8
Q

How does one generally separate proteins?

A

Chromatography.

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9
Q

What is ion-exchange chromatography?

A

Separates on the basis of charge.

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10
Q

What is gel-filtration chromatography?

A

Separates on the basis of hydrodynamic volume.

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11
Q

What is affinity chromatography?

A

Separates based on the affinity of a protein to the matrix.

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12
Q

How does one know where the protein of interest is after chromatography?

A

An assay that tests for specific activity (such as a Bradford assay).

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13
Q

How does immunoprecipitation work?

A

1) Grind up cells in extraction buffer, centrifuge to obtain total cell extract.
2) add antibody against specific protein.
3) add beads coupled to protein A (recognizes all antibodies)
4) centrifuge and keep pellet (contains beads-antibody-specific protein)

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14
Q

How does co-immunoprecipitation work?

A

Same as immunoprecipitation except use a milder buffer to not prevent protein-protein interactions.
Proteins that are associated with the immunoprecipitated protein will precipitate as well.
Identify with western blot or mass spec.

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15
Q

How does one genetically engineer tags for protein purification?

A 
Take gene for protein of interest.
Insert DNA encoding peptide epitope tag.
Introduce into a cell.
Cell makes an epitope-tagged protein.
Rapid-purification of tagged protein and any associated protein is accomplished. (Co-Ip)
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16
Q

Using SDS-Page, how does one identify proteins?

A

Seperate based on size. Incubate and place on membrane, add antibodies, determine proteins.

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17
Q

What is mass spectrometry?

A

1) Proteins are broken up into peptides.
2) Mass of peptide can be precisely determined..
3) Computer software can determine what combination of amino acids could give that mass.

18
Q

What is tandem mass spec?

A

Two steps:

1) Separate out a single peptide.
2) Break up into smaller peptides and determine mass of each
3) Determine aa sequence and can identify altered amino acids (ex: phosphorylated)

19
Q

How can protein function be selectively disrupted?

A

Through the use of drugs or small molecule inhibitors, through antibody depletion or genetically. (Genetically implies many techniques that lead to protein disruption, through mutations and other means)

20
Q

What is antibody depletion.

A

Use specific antibodies, mix with proteins and can selectively remove protein from mixture.

21
Q

What is the best method to analyze and manipulate DNA

A

Through synthesis in heterologous system.

22
Q

What is involved in DNA cloning?

A

Clone gene expression vector and synthesize protein of interest in heterologous system, then purify protein.

23
Q

What are the different studies that can be accomplished after protein purification and isolation from a heterologous system?

A

In vitro assays, test for direct interaction between two proteins with GST pulldown, determine protein structure, antibody production.

24
Q

What is GST pulldown?

A

Variation on affinity chromatography.
Express protein of interest in vector with GST gene.
This makes a fusion protein with GST which has a very high affinity for glutathione.
Purify from other proteins by passing lysate over glutathione beads.
Can use GST proteins to determine which proteins interact with protein of interest.
If they interact directly, these will precipitate together.

25
Q

How does one produce proteins in large amounts?

A

Clone gene into expression vector, grow in heterologous system, exploit enhancer to make abundance of protein. Cause overexpression.

26
Q

What are the different things that can be done with a protein from mass spec. identification?

A

Search database to get DNA sequence, make primers, PCR amplify, put in bacterial expression vector to get a lot of the protein, ca do biochemical tests to get more information on the protein such as structure, associated proteins, etc..

27
Q

How can protein structure be determined?

A

Through X-ray crystallography.

28
Q

How does X-ray crystallography work?

A

Start with protein of interest in solution.
Evaporate water from protein to form crystals (proteins arranged in same way)
Shoot X-rays through the protein crystals.
Get defined refraction pattern to determine 3D shape.

29
Q

What is NMR?

A

Does not require crystallization, works for smaller proteins. Method to determine 3D structure.

30
Q

What is a BLASTp search?

A

identify related proteins in same or other species.

31
Q

What is domain prediction?

A

identify putative domains based on sequence similarity to characterized domains.

32
Q

How does one study proteins with microscopy?

A

Can label protein of interest and see where they localize.

33
Q

How does one generate antibodies?

A

Purified protein obtained from bacterial expression (in vitro translation).
Alternatively, can do in vitro synthesis of a short peptide corresponding to part of the protein.
Injected into rabbit, mouse or other.
After several weeks obtain serum.
Serum contains antibodies against multiple epitopes on protein and non-specific antibodies (including fusion tag specific antibodies)
Specific antibodies can be purified by affinity purification.

34
Q

How does one produce monoclonal antibodies?

A

These are generally obtained from mice.
After immunization, remove spleen, harvest antibody secreting B-cells.
Fuse these to myeloma (cancer) cells, this produces a hybridoma whose cells can infinitely divide and produce a single antibody.
From the multiple hybridoma cell lines, identify the one that makes an antibody specific to the injected protein.
Supernatant from this cell line contains pure mono-specific antibodies.

35
Q

How are antibodies used to detect specific molecules?

A

Can recognize protein of interest through fluorescent immunolocalization. The expressed antigen is recognized by primary antibody, incubate, add secondary antibody which is fluorescent.

36
Q

How is the secondary antibody obtained?

A

Inject primary antibody in different animal, new animal makes antibodies against primary antibody.

37
Q

Why are secondary antibodies used?

A

These are generally bought.
Better for co-localization.
Also can amplify signal because secondary antibodies can recognize more sites on primary antibody.

38
Q

How can individual proteins be tagged in living cells?

A

GFP fusion protein.

39
Q

How can specific macromolecules be localized in the cell?

A

Immunogold electron microscopy.

40
Q

What is Immunogold electron microscopy?

A

Gold particles are attached to secondary antibody.

Can do double labelling, to distinguish by size of particles.

Molecular Cell Biology (9 decks)

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