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