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Cell Biology > Lecture 4 Slides > Flashcards

Lecture 4 Slides Flashcards

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0
Q

What is a tag

A

A peptide incorporated into the protein of interest

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1
Q

Why tag a protein

A

To determine location of protein in cell

To purify the protein and its associated proteins

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2
Q

What to use as tags

A

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

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3
Q

HA tag

A

Epitope of nine amino acids

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4
Q

GFP

A

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

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5
Q

How is GFP placed in target

A

Translational fusion of its coding sequence with gene of interest

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6
Q

How big is GFP

A

238 aa

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7
Q

Where does GFP come from

A

A jellyfish

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8
Q

Why does GGP fluoresce

A

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

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9
Q

Is there a variety to GFPs

A

Genetically engineered GFP is available in different colors

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10
Q

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

A

May not fold properly

May not display normal function or normal localization

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11
Q

How do you solve problems caused by using tagged proteins

A

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

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12
Q

Gold standard for showing tagged protein is functional

A

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.

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13
Q

What other function can GFP have

A

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

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14
Q

Explain pull down of proteins to study protein-protein interactions

A
  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
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15
Q

Glutathione

A

Tripeptide reducing agent in cells

16
Q

Glutathione-S-transferase

A

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

17
Q

Co-immunoprecipitation process

A
  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
18
Q

Technique for studying protein interactions in vivo

A

Yeast two-hybrid system

19
Q

What is GaI4 protein

A

A gene activator

20
Q

How many domains does GaI4 have. What are they.

A

Two.
DNA binding domain
Transcriptional activation domain

21
Q

How is galatokinase gene activated

A

Two GaI4 domains must interact to activate transcription

22
Q

Describe yeast two hybrid system

A

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)

23
Q

Wavelength of visible light

A

0.4-0.7 micrometers

24
Q

Resolution

A

Smallest distance between objects that appear distinct

25
Q

Limit of to resolution with light microscope

A

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

26
Q

Bright field light microscopy

A

Image obtained by light waves passing through specimen

27
Q

Phase contrast and no arks differential interference contrast (DIC)

A

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

28
Q

Dark field microscopy

A

Detects only the light scattered by components in the cell

29
Q

Fluorescent microscopy

A

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

30
Q

How are different colors of image combined

A

With imaging software

31
Q

Indirect immunofluorescence microscopy process

A
  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
32
Q

Fluorescence resonance energy transfer (FRET)

A
  • 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
33
Q

Super resolution microscopy

A

Involves new technology with microscope, image processes, and fluorophores that can be turned on and off

Allows resolution to ~50 nm

34
Q

Confocal microscopy

A

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.

35
Q

Transmission electron microscopy process

A
  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)
36
Q

Immunogold labeling

A

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

37
Q

Scanning electron microscopy (SEM)

A

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

38
Q

EM tomography -advanced TEM method

A

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

Cell Biology (35 decks)

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