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?)
2. Where are they?
3. Which are interacting with which?
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 protein
Get 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 conditions
2. ‘Fix’ cells in a tissue sample
Done with ‘formalin’ (formaldahyde, water,
methanol). Crosslinks proteins by forming
methylene bridges
Embed 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
fused
Myeloma cells
HGPRT-
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…
Streptavidin
Biotinylated
Horseradish
Peroxidase
2° Antibody
H2O2
Immunohistochemistry Examples
So histology and immunohistochemistry can tell us which
cells… Are producing how much protein
Prion Protein
PAX5
CD3, CD20
Beta III tubulin
(neurone specific)
HBcAg
NIH 3T3
In Situ Hybridization
But, what if we can’t make an antibody or the target protein
is inaccessible?
cRNA w/ probe
Target Protein
mRNA
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 Processing
For Staining
For Cutting
(microtome)
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
Restriction Enzyme site
Promoter
P
Target protein
Stop
Target
Linker (poly-G)
P
Same promoter = same level of
production!?
EGFP
Target
In Cell Localization: Fluorescence
Fluorescence/Immunohistochemistry is the most commonly
used tool to localize proteins at the sub-cellular level.
Actin
endoG-YFP
(apoptotic
endonuclease)
Mitochondria
Debrin
Colocalization
Apoptosis 12 (7): 1155-1171, 2007
Instrumentation: Confocal Microscopy
In 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!
Nitrocellulose Membrane
2°
Electrophoresis
All extract proteins
on membrane
(skim)
1°
Outside the cell: Antibody Microarrays
If 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 genes
Roundworm Genome?:
~ 20,000 genes
(admittedly, we humans
do more with our genes
than the roundworm via
RNA splicing)
Uncovering Protein/Protein Interactions
One of the first methods for uncovering Protein/Protein
interactions was the ‘yeast-two-hybrid’ screen
Any method used must be
parallel
Analyte proteins are
overexpressed with Gal4 AD and
BD UAS Promoter binders
Must be able to get into the
yeast nucleus
Weak, transient interactions
can still activate reporter
Consequently, 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…
Science 295 No.5553 (2002): p321-4
Phage Display
PD = 369 Interactions
Y2H
59 Interactions
Y2H = 233 Interactions
Protein Microarrays
In protein microarrays, proteins are ‘printed’ (literally) onto
a glass slide…
This microarray has
every protein in the
S. Cerevisiae genome
Proteomics (2003); 3(11):2190-9.
A ‘liver protein’
microarray
Proteomics 7 (13):
2151-2161 2007
Proteins are detected in ‘duplicate
spots’ to limit false positives
Protein Complementation
Protein complementation is the least versatile protein
interaction detection technique, but it may be the coolest…
Proteomics 7 (7):
1023-1036, 2007
Nat. Meth. 4 (5): 421-427,
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