Lecture 4 Slides Flashcards
What is a tag
A peptide incorporated into the protein of interest
Why tag a protein
To determine location of protein in cell
To purify the protein and its associated proteins
What to use as tags
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
HA tag
Epitope of nine amino acids
GFP
Green fluorescent protein tag is naturally fluorescent
No fixation and antibody required
Allows visualization of tagged protein in living cells
How is GFP placed in target
Translational fusion of its coding sequence with gene of interest
How big is GFP
238 aa
Where does GFP come from
A jellyfish
Why does GGP fluoresce
A natural fluorophore forms from three amino acids in the folded protein
Is there a variety to GFPs
Genetically engineered GFP is available in different colors
Potential problems with analyzing function/localization of a protein using a tagged version
May not fold properly
May not display normal function or normal localization
How do you solve problems caused by using tagged proteins
Place the tag at different positions of the gene; compare tag at N-terminus and at C-terminus of protein
Gold standard for showing tagged protein is functional
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.
What other function can GFP have
It can report promoter activity if it is transcriptionally fused with promoter of interest. GFP will show cells in which promoter is active
Explain pull down of proteins to study protein-protein interactions
- Construct a fusion gene:
Glutathione-S-transferase coding sequence fused to coding sequence for protein of interest - Express fusion gene in E.coli to obtain GST-fusion protein
- Mix cell extract with fusion protein; apply mixture to glutathione affinity column
- Fusion protein binds to column along with any interacting proteins
- Elite protein complexes from column using glutathione
Glutathione
Tripeptide reducing agent in cells
Glutathione-S-transferase
Is 26 kDa enzyme that catalyzes the conjugation of reduced glutathione to various;high affinity for glutathione
Co-immunoprecipitation process
- Generate an antibody against protein of interest
- Mix antibody with cell extract
- Add protein A beads (protein A binds IgG molecules). Centrifuge to collect beads.
- Wash beads to elute unbound proteins
- Boil beads in SDS-PAGE sample buffer to elute antibody plus protein complex
- Analyze proteins by SDS page
Technique for studying protein interactions in vivo
Yeast two-hybrid system
What is GaI4 protein
A gene activator
How many domains does GaI4 have. What are they.
Two.
DNA binding domain
Transcriptional activation domain
How is galatokinase gene activated
Two GaI4 domains must interact to activate transcription
Describe yeast two hybrid system
Two fusion genes are prepare, each carried on a plasmid transfection into yeast cells.
- One gene encodes GaI4 DNA binding domain + “bait” protein
- Second gene encodes GaI4 activation domain + “prey” protein
If bait and prey proteins interact, GaI4 protein will activate transcription of reporter gene (Lac Z)
Wavelength of visible light
0.4-0.7 micrometers
Resolution
Smallest distance between objects that appear distinct
Limit of to resolution with light microscope
~200 nm due to properties of light and limitations of lens construction. A smaller object might be detected it it emits light.
Bright field light microscopy
Image obtained by light waves passing through specimen
Phase contrast and no arks differential interference contrast (DIC)
Phase of light waves are shifted by passing through different cell components to generate contrast
Dark field microscopy
Detects only the light scattered by components in the cell
Fluorescent microscopy
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
How are different colors of image combined
With imaging software
Indirect immunofluorescence microscopy process
- Treat cells with nonionic detergent to permeabilize membranes and with fixative to cross link proteins
- Incubate cells with primary antibody against antigen
- Wash away unbound antibodies
- Incubate with secondary antibody against primary antibody. Secondary antibody is commercially available and amplifies signal.
- Wash away unbound antibodies
- View secondary antibody with fluorescence microscope
Fluorescence resonance energy transfer (FRET)
- 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
Super resolution microscopy
Involves new technology with microscope, image processes, and fluorophores that can be turned on and off
Allows resolution to ~50 nm
Confocal microscopy
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.
Transmission electron microscopy process
- Tissue/cell samples are fixed to cross link and stabilize proteins and lipids
- alternative to chemical fixation is freezing - Specimens are dehydrated; then embedded in plastic resin. Sections 50-100 nm are cut.
- Staining with heavy metals such as Pb allow visualization of cell organelles
- Beam of electrons passed through specimen allows high resolution (at least 100 times higher than light microscope)
Immunogold labeling
Antibody treatment during electron microscopy labeled with gold particle, which will be detected as a round dot
Scanning electron microscopy (SEM)
Specimen (cell, organelle, membrane) is coated with heavy metal to give 3D effect.
EM tomography -advanced TEM method
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
