KH7

Question 1

What is chromatography

Answer 1

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

Question 2

What is the movement of the mobile phase in chromatography

Answer 2

Liquids tor gases move continuously past the solid phase

Question 3

For protein work, the mobile phase is generally

Answer 3

A liquid, an aqueous buffer

Question 4

Characteristics of the rate of movement of protein molecules in chromatography

Answer 4

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

Question 5

What are different chromatographic methods based on

Answer 5

Different kinds of interactions of the proteins with the solid phase

Question 6

Physical structure of chromatography

Answer 6

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

Question 7

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

Answer 7

Size

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

Question 8

How are small proteins filtered in gel filtration chromatography

Answer 8

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

Question 9

How are large proteins filtered through gel filtration chromatography

Answer 9

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

Question 10

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

Answer 10

Electric charge

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

Question 11

What is an antibody and what does it do

Answer 11

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

Question 12

How many epitopes can each antibody molecule recognize

Answer 12

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

Question 13

How does antibody affinity chromatography work

Answer 13

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

Question 14

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

Answer 14

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

Question 15

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

Answer 15

Lowering the pH

Question 16

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

Answer 16

The Fc portion

Question 17

What is a characteristic of the Fc portion of an antibody

Answer 17

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

Question 18

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

Answer 18

The Fab portion

Question 19

What part of the antibody varies among antibodies within a species

Answer 19

The Fab portion

Question 20

How can a protein complex be isolated from a protein mixture

Answer 20

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

Question 21

What is immune precipitation

Answer 21

Recovery of protein complexes that bind to a specific antibody

Question 22

How is the antibody:antigen recovered in immunoprecipitation

Answer 22

Precipitation

Question 23

What will the immunoprecipitate contain

Answer 23

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

Question 24

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

Answer 24

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

Question 25

What is the co-immunoprecipitation experiment steps

Answer 25

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

Question 26

What is the conclusion of PPARalpha

Answer 26

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

Question 27

What is immunofluorescence microscopy

Answer 27

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

Question 28

What does indirect immunofluorescence involve

Answer 28

Antibodies raised against the constant region of antibodies

Question 29

How are the antibodies in immunofluorescence microscopy made

Answer 29

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

Question 30

What permits detection in fluorescent microscope

Answer 30

Chemical coupling of a fluorochrome to the secondary antibody

Question 31

Steps of immunofluorescence microscopy

Answer 31

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

Question 32

What is the purpose of double-label immunoflurescence

Answer 32

Simultaneously observe two proteins

Question 33

What is green fluorescent protein (GFP)

Answer 33

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

Question 34

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

Answer 34

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

Question 35

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

Answer 35

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

Question 36

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

Answer 36

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

Question 37

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

Answer 37

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

Question 38

How would actin or alpha tubulin be detected in western blots

Answer 38

Using an antibody raised against it

Question 39

How would we use a western blot to find an Tmem65

Answer 39

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

Question 40

What is the difference between immunoprecipitation and coimmunoprecipotation

Answer 40

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

Question 41

What is the immunoprecipitate

Answer 41

Everything that reacts with the antibody