Lecture 5: Cellular Level Methods. So far we’ve seen some methods for assessing the chemical and/or physical state of a protein.
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
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
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
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
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.
Monoclonal antibodies are made by injecting an animal with your target analyte.
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
One of the first, and currently most commonly used detection systems is…
So histology and immunohistochemistry can tell us which cells…
Are producing how much protein
Beta III tubulin (neurone specific)
But, what if we can’t make an antibody or the target protein is inaccessible?
cRNA w/ probe
Target Protein mRNA
In Situ Hybridisation is a little more specific, allowing us to quantitate within cells, but mostly still used at tissue level
For Cutting (microtome)
To make a Chimera, the gene encoding the protein of interest is modified to encode the analyte plus the reporter
Restriction Enzyme site
Same promoter = same level of production!?
Fluorescence/Immunohistochemistry is the most commonly used tool to localize proteins at the sub-cellular level.
endoG-YFP (apoptotic endonuclease)
Apoptosis 12 (7): 1155-1171, 2007
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).
‘Western blots’ are basically Immunohistochemistry outside the cell
Bust it open!
All extract proteins on membrane
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.
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:
(admittedly, we humans do more with our genes than the roundworm via RNA splicing)
~ 20,000 genes
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 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
Both Phage Display and Yeast Two Hybrid can produce extremely complicated interaction maps, if the genome is well known
But both these techniques have high rates of false positives, so…
Science 295 No.5553(2002): p321-4
PD = 369 Interactions
Y2H = 233 Interactions
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 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
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