Lecture 5: Cellular Level Methods So far we’ve seen some methods for assessing the chemical and/or...
-
Upload
grant-mckinney -
Category
Documents
-
view
213 -
download
0
Transcript of Lecture 5: Cellular Level Methods So far we’ve seen some methods for assessing the chemical and/or...
Lecture 5: Cellular Level Methods
So far we’ve seen some methods for assessing the chemical and/or physical state of a protein.But those are fundamental questions. To get a ‘direct’ understanding of what these proteins are doing biologically, we need monitor them on the cellular level. We need to know…1. How much there is (response to
stimulus?)3. Which are interacting with which?2. Where are they?
These questions can be answered with:
(1 and 2) Histology/Microscopy
(3) Yeast two Hybrid, Complementation
How much is there?
Remember, most of the methods we’ve talked about for observing proteins are not quantitative. So no matter what happens, we are likely only going to be able to get a relative answer.Effectively, this will limit us to answering the ‘how much’ question for stimulated or ‘disease state’ versus ‘normal state’ cells.
But before we can do any of that, we need to:Get the label in and attached to the correct proteinGet the cells in a state where they can be observed
Getting the Label in: Histology
Very often, the molecular biology required to transform eukaryotic cells is prohibitive. An alternative option is ‘fix’ the cell at a certain time and then label it. This is cellular level histology.1. Grow cells under desired
conditions2. ‘Fix’ cells in a tissue sampleDone with ‘formalin’ (formaldahyde,
water, methanol). Crosslinks proteins by forming methylene bridgesEmbed cells in parafin wax.
3. Cut thin ‘slices’ of wax embedded tissue. Dry on to cover slip
Histology: Chemical Stains
You are now ready to attach your probe
The older probes are dyes that bind to specific regions or organelles for visualization:
H&E (Hematoxylin/Eosin) Stain
Wright Stain for Immune Cells
Histology: Immunohistochemistry
Chemical Stains are non-specific. They rarely target a specific protein. Immunohistochemistry uses modified antibodies to target specific proteins/molecules.Polyclonal antibodies are made by injecting an animal with your
target analyte.
Immune response
Monoclonal antibodies are made by injecting an animal with your
target analyte.
spleen cells
Myeloma cellsHGPRT- fuse
d
Immunohistochemistry
So these immunoglobulins will ‘stick’ to the antigens against which they are raised in our fixed cells. But how do we see them?Of course we’re going to add a chromophore. But why modify every antibody you make when you can create a generic ‘secondary antibody’ directed at the unchanging part of the ‘primary’ antibody:We can direct
antibodies at the ‘constant’ part of the heavy chain
Biotin/Strepavidin/HRP Detection
One of the first, and currently most commonly used detection systems is…
2° Antibody
Biotinylated
Streptavidin
Horseradish Peroxidase
H2O2
Immunohistochemistry Examples
Beta III tubulin (neurone specific)
PAX5
CD3, CD20
HBcAg
Prion Protein
So histology and immunohistochemistry can tell us which cells…
NIH 3T3
Are producing how much protein
In Situ Hybridization
But, what if we can’t make an antibody or the target protein is inaccessible?
Target Protein mRNA
cRNA w/ probe
In Situ Hybridization Examples
In Situ Hybridisation is a little more specific, allowing us to quantitate within cells, but mostly still used at tissue level
CD5 Chromosome 1
Histology Instrumentation
For Cutting (microtome)
For Processing
For Staining
Getting the Label In: Chimeras
To make a Chimera, the gene encoding the protein of interest is modified to encode the analyte plus the reporter
P TargetPromoter
Target protein
Stop
P TargetEGFP
Restriction Enzyme site
Same promoter = same level of production!?
Linker (poly-G)
In Cell Localization: Fluorescence
Fluorescence/Immunohistochemistry is the most commonly used tool to localize proteins at the sub-cellular level.Actin
Debrin
Apoptosis 12 (7): 1155-1171, 2007
endoG-YFP (apoptotic endonuclease)
Mitochondria
Colocalization
Instrumentation: Confocal MicroscopyIn confocal microscopy, the illuminating light is focused on a tiny section of the sample.
The primary advantage of confocal microscopy is that it eliminate any light that is not from the focal plane of the focusing lens (which would be out of focus).
Outside the Cell: The Western Blot
‘Western blots’ are basically Immunohistochemistry outside the cell
Bust it open!
Electrophoresis
Nitrocellulose Membrane
All extract proteins on membrane
(skim)
1°
2°
Outside the cell: Antibody MicroarraysIf you want to analyze the proteome in parallel…
This method is semi-quantitative. You can use a known concentration of antigen as a standard.
What Sticks to What: The ‘Interactome’One of the most pressing questions in biochemistry is protein function. You can tell a lot about what a protein does by figuring out what it interacts with.This – and not the gene level – is where the complexity of life arises:
Human genome?:20,000-25,000
genesRoundworm Genome?:
~ 20,000 genes
(admittedly, we humans do more with our genes than the roundworm via RNA splicing)
Uncovering Protein/Protein InteractionsOne of the first methods for uncovering Protein/Protein interactions was the ‘yeast-two-hybrid’ screenAny method used must be parallelAnalyte proteins are overexpressed with Gal4 AD and BD UAS Promoter binders Must be able to get into the yeast nucleusWeak, transient interactions can still activate reporterConsequently, Y2H screens are considered low confidence
Phage Display
Phage Display relies on the ‘display’ of a peptide sequence on the C-terminus of a phage coat protein (pIII, IV or 10B)These are made to interact with
a ‘library’ of immobilized proteins or peptides
Can use unnatural selection to amplify good binders
Phage Display and Yeast 2 Hybrid
Both Phage Display and Yeast Two Hybrid can produce extremely complicated interaction maps, if the genome is well known
Phage Display and Yeast 2 Hybrid
But both these techniques have high rates of false positives, so…
Phage Display
Y2H
PD = 369 Interactions
Y2H = 233 Interactions
59 Interactions
Science 295 No.5553 (2002): p321-4
Protein Microarrays
In protein microarrays, proteins are ‘printed’ (literally) onto a glass slide…
This microarray has every protein in the S. Cerevisiae genome
A ‘liver protein’ microarray
Proteins are detected in ‘duplicate spots’ to limit
false positives
Proteomics 7 (13): 2151-2161 2007
Proteomics (2003); 3(11):2190-9.
Protein Complementation
Protein complementation is the least versatile protein interaction detection technique, but it may be the coolest…
Nat. Meth. 4 (5): 421-427, 2007
Proteomics 7 (7): 1023-1036, 2007
Time-Resolved Localization
Fluorescent labels can be used in living cells to monitor protein localization in real time.
Apoptosis 12 (7): 1155-1171, 2007 BBRC 364 (2): 231-237, 2007