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BIOL 200 > KH7 > Flashcards

KH7 Flashcards

1
Q

What is chromatography

A

Separation of components based on their differential interactions with an immobile (solid) material

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2
Q

What is the movement of the mobile phase in chromatography

A

Liquids tor gases move continuously past the solid phase

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3
Q

For protein work, the mobile phase is generally

A

A liquid, an aqueous buffer

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4
Q

Characteristics of the rate of movement of protein molecules in chromatography

A

Protein is moved along in the mobile phase but at rates that depend on how much they interact with the solid phase

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5
Q

What are different chromatographic methods based on

A

Different kinds of interactions of the proteins with the solid phase

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6
Q

Physical structure of chromatography

A

in columns with a solid phase made up with millimeter sized beads

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7
Q

What is the separation based on in gel filtration chromatography and how is it done

A

Size

Solid phase gel beads have pores of molecular dimensions, mobile phase flows between beads and freely enters and exits

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8
Q

How are small proteins filtered in gel filtration chromatography

A

They can enter and exit the beads through the pores, takes longer to filter

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9
Q

How are large proteins filtered through gel filtration chromatography

A

Large proteins can’t go through beads and so they run ahead in mobile phase, go fast

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10
Q

What is separation based on in ion exchange chromatography and how does it work

A

Electric charge

Protein molecules with a net electric charge will bind to the immobile phase having the opposite charge

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11
Q

What is an antibody and what does it do

A

A protein that recognizes, by highly specific binding, a molecular target (epitope) present on the antigen molecule against which the antibody was raised

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12
Q

How many epitopes can each antibody molecule recognize

A

One but many different antibodies can be raised for many different antigens

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13
Q

How does antibody affinity chromatography work

A

Antibodies specific for any particular protein can be covalently coupled to the solid phase

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14
Q

If a complex protein i true is flowed through a column in antibody affinity chromatography, what will be retained

A

Only the protein to which the antibody is able to bind to is retained in the column

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15
Q

How is the target protein released from the antibody once the antibody-affinity chromatography is complete

A

Lowering the pH

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16
Q

What is the region of the primary antibody that is recognized by the secondary antibody of another species

A

The Fc portion

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17
Q

What is a characteristic of the Fc portion of an antibody

A

Constant among antibodies within a decides but different from species to species

18
Q

What is the region of the primary antibody that recognizes the epitope of interest

A

The Fab portion

19
Q

What part of the antibody varies among antibodies within a species

A

The Fab portion

20
Q

How can a protein complex be isolated from a protein mixture

A

Using an antibody that is specific for one protein of the complex

21
Q

What is immune precipitation

A

Recovery of protein complexes that bind to a specific antibody

22
Q

How is the antibody:antigen recovered in immunoprecipitation

A

Precipitation

23
Q

What will the immunoprecipitate contain

A

The protein carrying the epitope recognized by the antibody and any partner proteins stably associated with that protein

24
Q

What do western blots of SDS-solubilized cells reveal in immunoprecipitation

A

Presence of glucocorticoid receptor GR and PPAR alpha in cells treated (+) or untreated (-) with GR ligand

25
Q

What is the co-immunoprecipitation experiment steps

A
  1. Non-SDS cell homogenize (natural, non-denatured proteins, bound to partners)
  2. Immunoprecipitate with anti-GR antibody
  3. Add SDS and do western blot looking for presence of PPARa in immunoprecipitate
26
Q

What is the conclusion of PPARalpha

A

It forms a stable complex with GR but only when the latter is bound to its ligand

27
Q

What is immunofluorescence microscopy

A

Studying the distribution of proteins at the cellular and sub cellular level using antibodies that specifically recognize the protein of interest

28
Q

What does indirect immunofluorescence involve

A

Antibodies raised against the constant region of antibodies

29
Q

How are the antibodies in immunofluorescence microscopy made

A

Inject purified antibodies into a rabbit, the rabbit recognizes the mouse antibodies as foreign and make antibodies against the mouse antibodies

30
Q

What permits detection in fluorescent microscope

A

Chemical coupling of a fluorochrome to the secondary antibody

31
Q

Steps of immunofluorescence microscopy

A
  1. Prepare sample and place on microscope slide
  2. Incubate with primary antibody, wash away unbound antibody
  3. Incubate with fluorochrome-conjugated secondary antibody, was away unbound antibody
  4. Mount specimen and observe in fluorescence microscope
32
Q

What is the purpose of double-label immunoflurescence

A

Simultaneously observe two proteins

33
Q

What is green fluorescent protein (GFP)

A

A single polypeptide chain that contains enzymatic activity that modifies some of its own amino acid side chains to generate fluorochrome

34
Q

What can GFP be used for once introduced in cells and ultimately reproduced

A
  1. Reporter gene for transcriptional control elements
  2. Made into fusion protein to study intracellular protein localization
35
Q

What is the result of the GFP coding sequence replacing the protein coding sequence of the ODR10 gene

A

GFP expressed only in cells where the ODR10 gene is transcription ally active = specific sensory neurons (long path)

36
Q

What is the result of GFP coding sequence fused to the protein coding sequence of the ODR10 gene

A

ODR10-GFP fusion protein is targeted to the sub cellular region to which ODR10 is normally targeted

37
Q

Why in antibody affinity chromatography does eluding with pH3 buffer remove bound protein from antibody

A

Protein unfolds a bit allowing for the antigen to let it go

38
Q

How would actin or alpha tubulin be detected in western blots

A

Using an antibody raised against it

39
Q

How would we use a western blot to find an Tmem65

A

Alternative splicing of a single gene to give multiple proteins of different sizes

40
Q

What is the difference between immunoprecipitation and coimmunoprecipotation

A

I: Only looking for target of antibody
CoI: Looking for protein complex (target of antibody and protein that binds to target)

41
Q

What is the immunoprecipitate

A

Everything that reacts with the antibody

BIOL 200 (13 decks)

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