Notes 2: Analysis of Gene Expression (protein level)

Fluorescence Microscopy

Here we go
that's how we label things for fluorescence microscopy
like I said before for white light microscopy in like histology, we use so many different types of dyes
I'm not even going to cover that
so we'll talk about fluorescence microscopy because that's what we use most these days
there are essentially 3 different types
anyway, immunofluorescence, fusion tagging and affinity binding

let's go one by one.

Production of Polyclonal Antibodies

Let's start with immunofluorescence.
In order to perform immunofluorescence, we need to produce antibodies
We're not gonna use our own antibodies, that's not very ethical
We're gonna use antibodies produced in animals
Let's say, for example, we've seen before we um we've seen collagen that was labeled, let's pretend it was labeled with an antibody
so we want to produce antibodies that very specifically recognize collagen alpha one.
It's a typical collagen one alpha one collagen type one alpha one chain. That's the whole name of the thing
So we purify collagen
we injected it usually into a rabbit, but it could be a goat, it could be a donkey, it could be a hamster, it could be any other animal
we inject it and then this poor animal is going to mount a response to that antigen effectively because we are injecting something that they've never seen before
Obviously, we're not going to inject rabbit collagen into a rabbit because a rabbit otherwise would not recognize it in its own self because rabbits have seen a lot of rabbit collagen already and they've already developed tolerance to rabbit collagen
So we will use rat collagen or we will use porcine collage anyway, collagen derived from a species that is not rabbit. Otherwise the rabbit will not mount an immune response
The animal mounts an immune response
the B cells are selected, they expand all what we went before and what is important to know here this, we call polyclonal antibodies
Why? Because we are injecting the whole collagen protein
A collagen chain is big is
type one, alpha one is somewhere around 90 kilodaltons or something like that if I remember
I can't remember the details. But anyway, it's a big protein and there will be lots of areas of that proteins that are taken up
the protein is taken up through the process that we know before
a lot of bits and pieces will be presented by the dendritic cells to the T helpers and then the B cells as well
we see lots of different bits and pieces of this antigen
each bit is called an epitope
several different clones of B cells will be selected, one will be selected by an epitope at the N terminus. One will be selected by an epitope at the C terminus, another one by an epitope somewhere in between and so on and so forth, dozens of different epitopes.
So dozens of different clones of B cells are selected and expanded and these dozens of cells, the rabbit will make memory b cells from these clones and also plasma cells which will secrete antibodies
so after a few weeks, get the blood from the rabbit, purify the antibodies, and the vast majority of the antibodies that we can get are these antibodies that the rabbit is produced against this specific antigen that we have injected unless we're unlucky and the rabbit also got a cold. And instead of getting the antibody from collagen, we get antibodies to whatever cold virus it got to avoid that
we can purify using an affinity purification, a column with collagen attached to it and then purify on that and purify only the entire collagen antibodies
so that's how we make polyclonal antibodies
by polyclonal we mean that we get all the antibodies recognize one portion of collagen and all of them
Each clone of this will recognize a specific epitope

Production of Monoclonal Antibodies

we can also make monoclonal antibodies
How do we make monoclonal?
the process is a little bit longer
Usually we use mice, but these days there are lots of monoclonals that are produced also in rats and rabbits, especially rabbits
People have developed protocols for rabbit monoclonal antibodies that work really well
So it starts the same injecting the antigen into the animal
The animal mounts an immune response
Now instead of looking at the B cells and getting the antibodies from the B cells
So from the serum, from the blood, we take the spleen
remember, the spleen is also peripheral lymphoid organ, we get spleen cells and some people developed a protocol where they can get myeloma cells.
this is a myeloma
It should be of the B cell lineage. Otherwise it doesn't produce antibodies.
So people have developed protocols to fuse the spleen cells with myeloma cells.
So in the end, what's gonna happen is that the spleen cells are going to give the myeloma cells the genetic information to produce certain antibodies
the antibodies that recognize the antigen that we have injected, not all the cells will fuse some will not fuse
so first we select the ones that are fused and then we expand the ones that are fused and we expand them in clones
So from this polyclonal mixture of cells, because each spleen cells will be expressing one different, it would be the equivalent of a different clone of B cells
So the VDJ or combination would have happened differently.
Then plated them like one cell per well and then we expand them and then at some point to induce them to produce and secrete antibodies and then we can select which of these antibodies is the best to recognize only and exclusively the antigen that we wanted to recognize and not cross react
In the case of collagen type one, alpha one, for example, not cross react with alpha two or with the alpha one chain of collagen type four and so on and so forth.
So the advantage is that we can collect antibodies that are very highly specific for a specific epitope, not for not a population of different antibodies that recognize different epitopes but only one, let's say the one that is square here that only recognizes the square epitope. So we can obtain large amounts of just that clone of antibodies. And so it's very highly specific and it doesn't cross react with other proteins that are similar but not identical.
because they are so pure, they're so specific. We can actually use them as therapy.
Like we saw last time in immunotherapy, the antibodies monoclonal antibodies against, for example PD-L1, PD1 or CTLA 4, that's how they're produced
so they're produced with this protocol, but within a manufacturing pipeline that is called GMP, which is um uh I think it means great medical products or something like that.
so that is really a clean
for injection into a person that there's no contaminants, there's notoxic, especially contaminants and things like that
there's little variability from batch to batch because once one clone is established as a clone, everything is clonal and they're gonna produce always the same antibody
Whereas with the polyclonal, we have to inject the antigen again and in the second batch might be slightly different from the previous batch. So you know, it can change.

Disadvantages, the signal produce can be lower because now we are detecting one epitope. So we only get one antibody per one copy of the protein.
Whereas with the polyclonal, each copy of the protein can be bound by several antibodies. And since each antibody, we'll see in a moment, we produce one quantum of signal. One thing is to have the same protein bound to many antibodies. So to many quanta signal
Another disadvantage is if only this epitope is being recognized, you only have one copy of antibody per copy of protein so the signal can be lower
Sometimes what you want to actually is polyclonal.
For example, we have a chicken project in the lab and there are very few antibodies to chicken cells. So we're trying a lot of polyclonal antibodies that have been developed using antigens from mice. And we're hoping that some of these antibodies in the polyclonal population recognized regions of that protein that are conserved across species. So not only recognize mouse, the mouse antigen that the antibodies have been produced to recognize, but we also cross react with the chicken antigen because it's a conserved region and it's present in mouse and in chicken as well.
So sometimes you want that dirty situation because you want to recognize more or for example, you want to see a protein that goes through a proteolytic processing. So you want to have a polyclonal antibody. So you can see the protein, the full length of the protein. But you can also see after it's cleaved once after it's cleaved twice and produces different fragments because you will have a population of antibodies that will recognize various portions of the protein. So we will give you a signal, for example, in a western blotting at various levels. So sometimes you want that. Um and, and, and with a monoclonal antibodies, you can't get that
the development phase of a monoclonal antibodies is antibodies much longer and more expensive.

Immuno Assays

immunoassays are the ones that use antibodies and use the power of the antigen antibody interaction, which is a very strong interaction
it's not covalent, but it's very strong
there are many different types of immunoassays
in an immunoassay. Basically, the concept is that there is a mixture of molecules, it could be a piece of tissue, it could be a protein extract, it could be serum from the blood, iso liquid, it could be anything.
There's a mixture of molecules, often proteins, but they don't have to be proteins. They could also be other kind of molecules as long as they're recognized and recognize the ball by an antibody.
Or I should say, as long as we can produce antibodies that recognize that molecule here, the molecule of interest is this green triangle. And then there are the squares and the circles that we're not interested in. We've purchased an antibody that is produced by the company by immunizing rabbits. Let's say it's a polyclonal antibody for mice. And then they've made a monoclonal doesn't really matter. In this context, we have an antibody that recognizes the green circles does not recognize the squares or the triangle. So you see the antibody that interacts very strongly with the green triangle, not very strongly with the circle or the square, we add the antibody to this mixture of proteins for some time after which we do a wash and we remove everything that is not strongly bound.
So what's left is what the antibody bound to the triangle because it's strongly bound.
What is gone is the antibodies that had very loosely interacted with squares and circles because the interaction wasn't strong enough
now at this point, we can follow two paths.
Either the antibody we've used is already conjugated to a fluorophore.
Remember the ones we saw before like this, these are the fluorophores that have these rings with the free electrons that once they are hit by a photon, they capture the photon and they emit blah blah blah.
So these are fluorophores a small molecules
so we can attach the fluorophores to the to the back of the antibody
we can see the signal under epifluorescent or confocal microscopy or we can use an imager if this is a western blotting or we can use a plate reader if it's analyzer.
There are many different ways or another way if what we call the primary antibody by primary, we mean the antibody that is recognized the molecule of interest the triangle here, in this case.
If the primary antibody is not directly conjugated to a fluorophore or it could be also a chromogen. So something that develops a color rather than a than fluorescent light
it doesn't really matter what the readout is.
So if the primary is not directly conjugated, all we need to know is in which species it was produced.
For example, it was produced in mouse.
Well, it turns out that while the variable region of the antibodies are very specific for the antigen that they're supposed to recognize the constant fragment, which is the stick of the Y that is species specific.
All human antibodies have the same, all mouse antibodies have the same, which is different from humans, different from rabbit, different from goat, different from bird, different from all the other species.
So they're specific to a species.
So what we can, what people have done, they have taken this constant fragment of, let's say a mouse antibody, they have expressed it and purified it and they've used that to immunize another species, let's say to immunize a rabbit or a goat with that mouse constant fragment.
So now the goat, let's say has produced antibodies that recognize the constant fragment of the mouse. So we call them anti-mouse antibodies. And those are the blue here that we call secondary antibodies.
So we have in the lab or in a diagnostic lab, a collection of anti-mouse, anti-rat anti rabbit.
So whichever the primary antibody is, we can recognize it with that secondary antibody that is directly conjugated to a fluorophore.
And usually these secondary antibodies are polyclonal.
So they recognize various regions within the constant fragment of a of a primary antibody.
So the signal gets amplified because now one primary antibody, one copy of the primary antibody, you get several secondarys stuck to it, each one with their fluorophore.
So the signal gets amplified and we can see it better under the microscope.

Immunoasays

so this is one way of tagging
in order to use this way, the specimen has to be dead, fixed and depending on what we're looking for, it might have to be permeabilized
by permeabilized. We mean that if we're looking something that is inside the cells, then we have to poke holes in the plasma membrane of the cells because antibodies are big molecules, they're about 150 kilodalton the whole tetrameter and they don't just diffuse through the membrane, we have to make them go in
This process of poking holes in the plasma membrane is called permeabilizations done with detergents
so, detergents are amphipathic, they have one area that is hydrophobic, one area that is hydrophilic, they make pores in the plasma membrane
they're big enough for antibodies to get through
so if we're looking for something inside the cell, we have to also permeabilize.

Fusion Tagging

If we want to visualize live cells, then we have to use fusion tagging
Fusion tagging is like in this case, for example, tubulin, the green one is being visualized through fusion tagging
these are still fixed and permeabilized samples because we're using an anti-actin antibody to see actin in red
But tubulin we could see it when the cells were still alive because we had generated a fusion protein where tubulin was fused to green fluorescent protein.

Fluorescent Proteins

So green fluorescent protein or the other fluorescent proteins are proteins expressed in marine organisms, mostly jellyfish, but there's a lot of plankton that are also fluorescent
and they have this beta barrel that spontaneously cyclize and forms a fluorophore with the electrons that goes all around
there are so many different variants in nature, they can be excited by different wavelengths of light and emit in different wavelengths
then in addition to the ones that already exist in nature, people in the lab through mutagenesis have generated even more
some of them dimerized, some of tetramerized like the DS thread
this is just a small sample of all the various fluorescent proteins that exist
people have been given stupid names like it all started with GFP, which is the green fluorescent protein. And E here stands for enhanced because it was mutagenize to be even brighter, let's say then it came YFP which is yellow fluorescent protein. Then people started giving names like banana, orange, tomato, strawberry and then they, you know, they're really going off um of the tangent plum raspberry, whatever
they all fluoresce in different wavelength as you can see here in this, in these tubes.

Fusion Tag Proteins

So how do we do that? We get the GFP cDNA?
Remember the cDNA is the coding DNA, all spliced and everything
we make a construct through ligation of the GFP attached to the cDNA of the protein of interest.
In this case, we have decided to attach it to the N terminus
we could attach it to the C terminus, we could attach it inside, it doesn't really matter what matters is what is the point of the experiment.
Then you decide where to attach the GFP.
and you make what is called a fusion protein, then transfect the fusion protein inside your cells.
Usually it's not like completely fused, there's a little linker so that the protein of interest can still fold normally without being bothered sterically by the GFP or RFP or M cherry M banana, whatever
then you just go to the microscope and look at it and this is I believe, actin labeled with GFP.