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Lecture 5: Cellular Level Methods. So far we’ve seen some methods for assessing the chemical and/or physical state of a protein.

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slide1

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

slide2

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

slide3

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

slide4

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

slide5

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.

Monoclonal antibodies are made by injecting an animal with your target analyte.

spleen cells

Myeloma cells

HGPRT-

Immune response

fused

slide6

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

slide7

Biotin/Strepavidin/HRP Detection

One of the first, and currently most commonly used detection systems is…

Streptavidin

Biotinylated

Horseradish Peroxidase

2° Antibody

H2O2

slide8

Immunohistochemistry Examples

So histology and immunohistochemistry can tell us which cells…

Are producing how much protein

Prion Protein

PAX5

HBcAg

Beta III tubulin (neurone specific)

CD3, CD20

NIH 3T3

slide9

In Situ Hybridization

But, what if we can’t make an antibody or the target protein is inaccessible?

cRNA w/ probe

Target Protein mRNA

slide10

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

slide11

Histology Instrumentation

For Processing

For Staining

For Cutting (microtome)

slide12

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

Stop

Target protein

Promoter

P

Target

Linker (poly-G)

P

EGFP

Target

Same promoter = same level of production!?

slide13

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

slide14

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).

slide15

Outside the Cell: The Western Blot

‘Western blots’ are basically Immunohistochemistry outside the cell

Bust it open!

Nitrocellulose Membrane

(skim)

Electrophoresis

All extract proteins on membrane

slide16

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.

slide17

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:

(admittedly, we humans do more with our genes than the roundworm via RNA splicing)

Human genome?:

20,000-25,000 genes

Roundworm Genome?:

~ 20,000 genes

slide18

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

slide19

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

slide20

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

slide21

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 = 233 Interactions

59 Interactions

Y2H

slide22

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

slide23

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

slide24

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