Lecture 4 Slides

Question 0

What is a tag

Answer 0

A peptide incorporated into the protein of interest

Question 1

Why tag a protein

Answer 1

To determine location of protein in cell

To purify the protein and its associated proteins

Question 2

What to use as tags

Answer 2

Short peptide epitope recognized by a commercially available antibody
Green fluorescent protein (GFP)
Peptide that bonds to a ligand - can be used to purify the protein on an affinity column

Question 3

HA tag

Answer 3

Epitope of nine amino acids

Question 4

GFP

Answer 4

Green fluorescent protein tag is naturally fluorescent
No fixation and antibody required
Allows visualization of tagged protein in living cells

Question 5

How is GFP placed in target

Answer 5

Translational fusion of its coding sequence with gene of interest

Question 6

How big is GFP

Answer 6

238 aa

Question 7

Where does GFP come from

Answer 7

A jellyfish

Question 8

Why does GGP fluoresce

Answer 8

A natural fluorophore forms from three amino acids in the folded protein

Question 9

Is there a variety to GFPs

Answer 9

Genetically engineered GFP is available in different colors

Question 10

Potential problems with analyzing function/localization of a protein using a tagged version

Answer 10

May not fold properly

May not display normal function or normal localization

Question 11

How do you solve problems caused by using tagged proteins

Answer 11

Place the tag at different positions of the gene; compare tag at N-terminus and at C-terminus of protein

Question 12

Gold standard for showing tagged protein is functional

Answer 12

Transform the tagged gene into a cell containing a mutant version of the gene. Expression of tagged protein should “rescue” the mutant phenotype to wild type.

Question 13

What other function can GFP have

Answer 13

It can report promoter activity if it is transcriptionally fused with promoter of interest. GFP will show cells in which promoter is active

Question 14

Explain pull down of proteins to study protein-protein interactions

Answer 14

1. Construct a fusion gene:
   Glutathione-S-transferase coding sequence fused to coding sequence for protein of interest
2. Express fusion gene in E.coli to obtain GST-fusion protein
3. Mix cell extract with fusion protein; apply mixture to glutathione affinity column
4. Fusion protein binds to column along with any interacting proteins
5. Elite protein complexes from column using glutathione

Question 15

Glutathione

Answer 15

Tripeptide reducing agent in cells

Question 16

Glutathione-S-transferase

Answer 16

Is 26 kDa enzyme that catalyzes the conjugation of reduced glutathione to various;high affinity for glutathione

Question 17

Co-immunoprecipitation process

Answer 17

1. Generate an antibody against protein of interest
2. Mix antibody with cell extract
3. Add protein A beads (protein A binds IgG molecules). Centrifuge to collect beads.
4. Wash beads to elute unbound proteins
5. Boil beads in SDS-PAGE sample buffer to elute antibody plus protein complex
6. Analyze proteins by SDS page

Question 18

Technique for studying protein interactions in vivo

Answer 18

Yeast two-hybrid system

Question 19

What is GaI4 protein

Answer 19

A gene activator

Question 20

How many domains does GaI4 have. What are they.

Answer 20

Two.
DNA binding domain
Transcriptional activation domain

Question 21

How is galatokinase gene activated

Answer 21

Two GaI4 domains must interact to activate transcription

Question 22

Describe yeast two hybrid system

Answer 22

Two fusion genes are prepare, each carried on a plasmid transfection into yeast cells.

1. One gene encodes GaI4 DNA binding domain + “bait” protein
2. Second gene encodes GaI4 activation domain + “prey” protein

If bait and prey proteins interact, GaI4 protein will activate transcription of reporter gene (Lac Z)

Question 23

Wavelength of visible light

Answer 23

0.4-0.7 micrometers

Question 24

Resolution

Answer 24

Smallest distance between objects that appear distinct

Question 25

Limit of to resolution with light microscope

Answer 25

~200 nm due to properties of light and limitations of lens construction. A smaller object might be detected it it emits light.

Question 26

Bright field light microscopy

Answer 26

Image obtained by light waves passing through specimen

Question 27

Phase contrast and no arks differential interference contrast (DIC)

Answer 27

Phase of light waves are shifted by passing through different cell components to generate contrast

Question 28

Dark field microscopy

Answer 28

Detects only the light scattered by components in the cell

Question 29

Fluorescent microscopy

Answer 29

Objects smaller than the limit of resolution may be detected if they emit light Fluorophore a absorb light at one wavelength and emit light at a specific longer wavelength

Question 30

How are different colors of image combined

Answer 30

With imaging software

Question 31

Indirect immunofluorescence microscopy process

Answer 31

1. Treat cells with nonionic detergent to permeabilize membranes and with fixative to cross link proteins 2. Incubate cells with primary antibody against antigen 3. Wash away unbound antibodies 4. Incubate with secondary antibody against primary antibody. Secondary antibody is commercially available and amplifies signal. 5. Wash away unbound antibodies 6. View secondary antibody with fluorescence microscope

Question 32

Fluorescence resonance energy transfer (FRET)

Answer 32

- to study interactions of two proteins in vivo - fluorescence emission from excited donor fluorophore overlaps with excitation peak of acceptor fluorophore - if donor and acceptor are adjacent (within 10 nm), FRET occurs

Question 33

Super resolution microscopy

Answer 33

Involves new technology with microscope, image processes, and fluorophores that can be turned on and off Allows resolution to ~50 nm

Question 34

Confocal microscopy

Answer 34

Uses a "pinhole" to reduce out-of-focus emitted light. Adjacent sections of the sample are imaged. The combined images have reduced background, increased resolution.

Question 35

Transmission electron microscopy process

Answer 35

1. Tissue/cell samples are fixed to cross link and stabilize proteins and lipids - alternative to chemical fixation is freezing 2. Specimens are dehydrated; then embedded in plastic resin. Sections 50-100 nm are cut. 3. Staining with heavy metals such as Pb allow visualization of cell organelles 4. Beam of electrons passed through specimen allows high resolution (at least 100 times higher than light microscope)

Question 36

Immunogold labeling

Answer 36

Antibody treatment during electron microscopy labeled with gold particle, which will be detected as a round dot

Question 37

Scanning electron microscopy (SEM)

Answer 37

Specimen (cell, organelle, membrane) is coated with heavy metal to give 3D effect.

Question 38

EM tomography -advanced TEM method

Answer 38

Sample is cryo-fixed with ultra rapid freezing to prevent formation of ice crystals. Sections are viewed with a high voltage electron microscope Specimen holder is tilted in the microscope to obtain views from different angles Computational methods used to create 3D reconstruction of specimen