- [Voiceover] Our project was done by
Bernard, Pengning, Christina, and Nathaniel
about affinity chromatography.
- Affinity chromatography specifically refers
to the laboratory technique used to separate mixtures.
Affinity chromatography is an idea centered
on the liquid chromatography by means
of reversible interactions between
biologically related agents and
target biological macromolecules.
The first example of affinity chromatography
to be documented was developed by Emil Stackenstein in 1910.
He utilized the natural interactions between
the enzyme alpa-amylase and a substrate starch
to create a column that bound alpha-amylase
to insoluble starch.
Not only was this the first example
of affinity chromatography, but also
one of the earliest examples of a separation
in which liquid chromatography was used
with an enzyme or a protein.
- As the next two decades passed by,
a major issue arose with the support
of what we know as a matrix.
Though the support was easy to prepare,
the binding properties were not long-term,
due to gradual loss of the binding agent.
A critical advancement took place in 1951
when Campbell, Luesher, and Lerman
immobilized bovine serum albumin or BSA
to an activated form of cellulose.
With their results, they were able
to isolate anti-BSA antibodies
in a serum sample from rabbits
that had previously been injected
and immunized with BSA.
- The next critical developments occurred in the mid-1960s.
One of them was created by Hjerten in 1964,
with the development of beaded agarose.
This was important because this support
was more readily adaptable to the cellulose
when proteins and bio-polymers
were used for liquid chromatography.
The next development happened in 1967,
when a group of scientists developed a method
known as cyanogen bromide immobilization method.
This method allowed for a general and convenient means
for combining peptides and proteins
to polysaccharide base materials.
Then a critical point happened in 1968,
when another group of scientists
coupled the beaded agarose development
along with the cyanogen bromide immobilization method.
This immobilized nuclease inhibitors to beaded agarose
and allowed for several enzymes to be purified.
By this new coupled method, the term
affinity chromatography was birthed.
- For our histogram, the number of publications
using the term affinity chromatography increased
rapidly after 1968.
There are still around 13 hundred
affinity chromatography papers
published during the last decade.
Now when the widely spread concept
of affinity chromatography, diversity in affinity ligands,
immobilization methods, and application
provides inspirational fields of scientific research.
We will look at some people who are using
affinity chromatography right here, in NC state.
- [Interviewer] Please introduce yourself.
- Hello, my name is Trino.
My last name is Ascencio-Ibanez.
I come originally from Mexico.
I've been in NC State for about 16 years,
since 2001, in different capacities.
My favorite thing to do here is teach,
but I also do research.
I love teaching, myself.
And I am the molecular and structural biochemistry
department, which is a really long name.
It's an awesome department,
and we do a lot of work with proteins.
- My main interest is in
what is the interaction between a pathogen and a plant.
I work with the virus that invade plants
of a specific family.
They are single strand of DNA viruses.
And they are called gemini viruses
because when you look at them in a electron microscope,
they really look like geminated particles,
and gemini means twins.
So they are called gemini viruses
because of that reason.
Well I am extremely interested in the cell cycle
and what is the host response to the viruses.
So I work in everything from understanding
how the virus infects to how the plant responds to it
and produce fats that are resistant to the viruses.
Well I use affinity chromatography
for several different purposes in my research.
One of the things that we have done
is we could used a plasma, we inserted a viral gene
in a bacterial plasma, and we modify it to add
some histogen tails that will be easy
to dig out when you put it in a complex mixture.
So the first thing with we did is was
we produced it in E coli,
and after the production we separated that
by using a nickel, nickel plates,
which separated whatever contained these histogens
from the rest of the proteinss.
And you do get some of the proteins that were not
what we were target proteins, but most of the thing
of them it was one of these viral proteins
that we were extremely interested in
producing antibodies with.
So we got the antibodies, those proteins,
and then we used them for immuno-histochemistry,
determination of where the virus was present.
Okay, so one of things that we use is a ricin
that contains nickel, and that it will have
high affinity to the histogens that we put
in the proteins that we are interested to dig out.
Then we go and put the ricin into a column like this,
and it will be variable that then some,
what's the amount of matter you're gonna be using,
it could be a finger or longer,
but you could also have very, very
thick columns this big, depends what you want to prove,
on the scale of your interest.
If you are looking for milligrams, micrograms,
or you're looking for grams of protein,
that's what will define this as well.
And also the source of your proteins,
as you're seeing bacterial egg sprouts,
you don't need so much, and we grow about
250, 500 mediators on bacteria.
Then we consolidate that, we make the lysis
on the bacteria and then we pass the output
from the column, and if the protein is present,
then nickel will work properly
and the buffer has proper pH,
then the protein will bind to the column.
Now then you can wash it.
And to dilute your protein, after washing,
you can also use different approaches.
You can use imidazol for instance,
that will exchange and make your protein come out
from the column.
But sometimes even changes in pH will
allow you to dilute the protein.
And you collect it, I don't have it here,
but you collect in a fraction collector,
otherwise you spend days here,
just dripping and counting drops
and closing tubes, which I have done.
But the thing is that it's really
a very interesting protocol
and one of the main things is that
you get high purity of the protein,
and that's something that we really like
to obtain from it.
- Hi, I'm Keith Weninger.
I am a professor of physics.
I've been at NC State for 12 years.
We do single molecule biophysics research.
We use tools of fluorescence microscopy
to look at protein-DNA interaction.
We attach fluorescent labels to proteins
through cysteine engineering.
And so we make lots of recombinant proteins
where we engineer cysteines at different locations.
And we express these in E coli
and we purify proteins with affinity chromatography.
We express proteins that have affinity tags,
most common are His tags to match with
nickel NDA matrix binding.
And we also use GST attached proteins
and glutathione media.
And so when the proteins are expressed in E coli
we lyse the cells and we
put it in soluble material and run it
over an affinity matrix to bind our protein of interest
and dilute it from the matrix after washing.
- [Interviewer] In this specific example,
what is your matrix?
- This is just an agarose, they're called NTA matrix.
- [Interviewer] Okay, and what's the ligand?
- [Professor Weninger] We are purifying DNA repair protein
from a different species that's expressed in E coli
and it contains a six-His signal tag.
- [Interviewer] So you said you're using
gravity flow here as opposed to, what are?
- This is, most of our work is just gravity flow columns,
wash it by hand and diluting in imidazol solutions.
- [Interviewer] You said imidazol solutions?
- Yeah, imidazol.
To release the six-His tag from the nickel NTA.
Once we purify the protein, we label it
with fluorescent dyes
and then we construct different experiments
in a fluorescence microscope,
where we look at interactions of these proteins
with DNA substrates.
- [Interviewer] So go ahead and introduce yourself.
- Hi, I'm Tony Planchart.
I'm an assistant professor of biological sciences.
- My research is involved in understanding how
the environment interplays with genetics
to cause human disease.
And we use animal models like the zebrafish
to better understand that.
But we also use cell culture models,
other in vitro models for that particular type of research.
Yeah, so one of the projects that we're working on right now
is to understand elements of the genome that are bound
by transcription factors and how those transcription factors
recognize the binding sites on the DNA.
So we will start by making a probe, a DNA probe
that has that sequence that we suspect
might be bound by a transcription factor,
and we'll conjugate it to a small molecule like biotin,
and then we will adhere that biotin label oligo
to a streptavidin column,
and then flow nuclear extract from different cells
or fish or fish lines that have been
either genetically manipulated or exposed
to different environmental compounds,
and look at the effect of these exposures
on the nuclear protein profile
and also see which ones bind to these particular sequences.
And then once we've done that,
we will cross-link the nuclear proteins
to these DNA fragments, and then we will wash them
off the column by adding excess biotin
so that we can displace them from streptavidin.
And then we will subject them
to proteomic analysis to identify the protein.
- [Interviewer] So this instrument here,
is that what you use for?
- This would be one type of instrument.
This is a low pressure chromatography instrument.
We will use this mostly for size exclusion chromatography.
But we can also use it for affinity chromatography as well.
- Hello, I'm Gongfang Hu.
I'm a fourth-year graduate student
working in Doctor Lindsey's lab,
department of chemistry, NC State University.
So here is our lab, which is kind of messy.
But this is a typical organic lab.
So here we do organic chemistry research
based on the porphyrin, which is the backbone
of the hemoglobin in your blood.
Yeah, so the story starts from the green leaves.
So you know green leaves harvest sunlight
and generate carbon, no sorry, generate oxygen
and the carbohydrates, we call them photosynthesis.
So there's a part of the research in chemistry
called artificial photosynthesis.
Here we artificially make some molecules,
especially organic or inorganic molecules.
They can do the same job as the green leaves,
like the carbon collect, and oxygen generation,
and carbohydrate generation, absorb those
carbon dioxide, or something like that.
That's my research focus on.
So here we design and synthesize
all kinds of large molecules,
including porphyrin, multi-porphyrin arrays
that can be applied to these kind of purpose.
So that's what we're doing.
But basically we design and synthesize molecules.
And then you characterize the molecule
with is it kind of for physical measurements,
like absorption spectrum, fluorescent spectrum
or something like that.
So that can tell you something.
Yeah, so the reason we use this is because,
you know, our target molecules become pretty large,
which has a molecular weight around 3,000 to 5,000.
That's pretty large for an organic molecule.
But the good thing is, the reaction to form
the target molecule is pretty simple.
You have two building blocks, building block molecules
which has a molecular weight like 15,000,
oh no sorry, 15 hundred, and a part of 15 hundred.
You combine them together, and boom, you got a 3,000 one.
So this is a mixture.
If you want to separate your entire product,
which has a 3,000 molecular weight
from your starting material or any other
kind of byproduct, they actually have a very different,
you know, in size, so that's why we can use
a size exclusion chromatography to separate them,
both the products or some residue of the starting material
or some byproducts or some over-produced material
or something.
But they are very different in molecular weight
or in size from the building blocks.
So that's why we use the SEC columns.
- [Interviewer] So can you run us through
a basic procedure using this
and explain what your matrix is,
the differences in these columns?
- Okay, so all of these are actually same type of things.
We bought these from the bio lab,
I don't know if you can see that, but no,
yeah that's a, the grade is actually
for different kind of rough solution,
some of the grade is higher, you can resolve
a larger molecule, but with a lower resolution.
But some of the grade is lower, you can only use
for very small, tiny molecules,
but the resolution is extremely high.
So the difference, they're all polymers
basically start with polymers.
They're cross-linked poly starter rings,
which means they're gonna form networks into the base.
But the difference of these columns,
they have a different size, the four sides
of the polymers are different.
So some of the molecules, if the molecule
is smaller than the four size,
it's gonna stuck in the molecule.
But if it's larger, it's gonna avoid getting into
the polymer, it's gonna leak through the edge
of the column, so which one, so it's gonna,
coming down from the edge of the column,
the wall of the column and come down pretty quick.
That's why, for typically SEC columns,
you can have a larger molecule go first
and smaller molecules go, you know,
in the background.