Hi there, I'm Jayme Dyer. This is a turtle, can you tell what sex it is? Yeah, me either.
But in a recently published paper, scientists identified a new way to determine the sex
of baby turtles
using a western blot which, it turns out, can be really helpful for conservation efforts.
Now, you might think that determining the sex of a baby turtle should be easy, right?
Just look up its skirt!
But looking at their genitals only works for sexually mature adult turtles. So I mean,
couldn't you just look at their sex chromosomes?
You probably know that for humans, females have two X chromosomes and males have an
X and a Y chromosome. So why not just analyze the sex chromosomes of the baby turtles?
Well, that won't work because most species of turtles don't have sex chromosomes. [ WHAT?!!
]
It turns out what determines whether a turtle becomes male or female is the temperature
of the nest when the eggs are incubating.
For most species, warmer nest temperatures result in females and cooler temperatures
result in males. This makes climate change especially problematic for endangered species
because as temperatures rise an imbalance in the male to female ratio can make it harder
for turtles to reproduce, which could lead to extinction.
So to try to save endangered turtle species scientists need a way to non-invasively monitor
the sex ratio of baby turtles on a large scale.
And the scientists who published this cool paper came up with a way to do that using
a western blot.
So, essentially, a western blot is just gel electrophoresis for proteins instead of DNA.
Now, if you don't know what gel electrophoresis is then the rest of this video probably won't
make sense, so go check out this video for more information. Okay, so a western blot
is a lot like gel electrophoresis. Scientists use an electric current to separate proteins
on a gel by size.
But to see the proteins in the gel they do something different than what they do for
a DNA gel.
Instead of using a dye that binds to DNA, they use antibodies that bind specifically
to a protein of interest to tell whether and how much of that protein is present in a sample.
So, the scientists studying baby turtles used a western blot to determine if a certain
protein was present in the blood of the baby turtles.
Even though their genitals aren't well developed yet, male and female turtles have different hormones
circulating in their blood. Now, not all hormones are proteins, but
one sex-specific hormone in turtles, called Anti-Mullerian hormone,
is a protein. Which means a scientist could detect it using a western blot.
By looking to see whether AMH was present in their blood, the scientists could tell
which baby turtles were males and which were females.
Here is the western blot from their paper. Interpreting a western blot is essentially
the same as interpreting a DNA gel from gel electrophoresis. Bands higher up in the gel
represent bigger proteins and fatter bands represent samples that have more protein.
But even though you interpret a western blot the same way you interpret gel electrophoresis,
HOW the bands were visualized is very different; and, to understand that difference you need
to understand how a western blot works.
There are three main steps to doing a western blot:
electrophoresis, transfer, and blotting. The first step, electrophoresis, is essentially
the same as gel electrophoresis except that it's called Sodium Dodecyl--Do--Dode--Dodecyl--Do--
- Computer: Dodecyl
- Jayme: Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis.
[ Wohoooo ]
It's usually just shortened to SDS-PAGE. It's just gel electrophoresis except instead of
making the gel out of agarose, which they do for DNA, they make the gel out of Polyacrylamide.
And the SDS part of SDS-PAGE comes from the name of the detergent that scientists add
to the protein sample before they load it on the gel. See, only negatively charged molecules
will move through the gel toward the positive pole,
but not all proteins are negatively charged.
So, scientists add SDS to the protein sample because SDS is negatively charged, and it
sticks
to all types of proteins and unfolds them. Making sure that each protein is a negatively
charged long chain of amino acids
that can snake through the pores of the gel.
Once the gel is done running you can't just see the proteins, they're tiny molecules.
Now, if you were running a DNA gel, at this point you would just add a dye that binds
to DNA and look to see where the bands are.
And actually, you can do that with an SDS page gel. It's called doing a Coomassie stain.
But it will show you all of the proteins in the sample, not just the one you're looking
for.
In the baby turtle study, the scientists ran a sample of blood on an SDS page gel,
and well, blood has a lot of proteins in it.
So, how do the scientists know which of these bands is the AMH protein?
That's where the next two steps of a western blot come in.
Remember that the goal of a western blot is to visualize specific proteins.
To do that, scientists use antibodies. Like literally the part of your immune system that
recognizes invaders.
It turns out that the mammalian immune system
is really good at creating protein antibodies that bind specifically to foreign proteins.
But where do scientists get an antibody that binds specifically to AMH protein?
Well, they injected a little bit of purified AMH protein into a mouse, or a rabbit, or
a goat.
For this paper, it was a mouse. So, that mouse mounted an immune response
and started making antibodies against AMH. Then scientists harvested those antibodies
and used them to visualize AMH protein in the western blot. But the problem is that
antibodies themselves are proteins, which means you can't just squirt them onto the
Polyacrylamide gel and hope that they can get in there.
And this is where the transfer step comes in. To get the proteins out of the gel, but
to keep them in this nice organized order,
scientists use an electric current to transfer the proteins from the gel to a thin plastic
membrane. Which holds the proteins in place while making them accessible to the antibodies.
Finally, we're at the blotting part of the western blot.
This is the point where the scientists squirt the antibodies on the membrane
to bind directly to AMH protein.
So, now, we have this membrane with antibodies stuck just
to the AMH protein, but you can't actually
see anything yet, right?
Antibodies are molecules, you can't see them by eye.
So we need a way to see where the antibodies are. For that, we use another kind of antibody.
We call the first one, the one that binds to
our protein of interest, a primary antibody.
Secondary antibodies bind to the primary antibody.
Literally, a biotech company injected a goat with mouse antibodies, and the goat produced
anti-mouse antibodies. But before selling those anti-mouse antibodies, the company modified
them either with a fluorescent molecule or an enzyme that converts
a chemical reagent into a colored product.
So, secondary antibodies give you a way to actually see where your protein of interest
is on the membrane.
And because multiple secondary antibodies bind to each primary antibody,
they help amplify the signal. Then all the scientists have to do is take a picture of
the membrane with a special camera. And finally, after all those steps, they can see their
western blot.
Now, you may be wondering why they call it a western blot, but it's actually a play
on words.
Back in the 70s, a scientist named Edwin Southern invented a method of detecting specific pieces
of DNA by running a gel and blotting afterward. Scientists called that method a southern blot,
named after Edwin. When scientists started running protein on gel and blotting for it
they called it a western blot. Actually, there's a northern blot too but not an eastern blot,
hmm.
Also just so you know, sometimes scientists will call a western blot an immunoblot or
immunoassay. The immuno referring to the antibodies that are used during the blotting step, but
they all mean the same thing.
So, the scientists who were studying the sex
of baby turtles, they used a western blot to
determine whether AMH protein was present in the baby turtle's blood. They found that
AMH protein was present in 100% of one-day-old male turtles but not in one-day-old female
turtles,
which they verified using laparoscopy to determine the sex of the turtles when they were older.
Having a non-invasive way to monitor sex ratios of baby turtles can be an important tool in
conservation efforts for endangered species.
All with the help of western blots.
Now, that's cool!