[Funky music] Thus far in lecture, we’ve been talking about
membrane proteins and the types that are possible within a lipid bilayer. However, what if you
don’t know what type of membrane protein you have. This animation is here to show you one
type of technique we can use to determine if a membrane protein faces the extracellular
side of the cytosol or the cytosolic side, and whether or not it can be a glycoprotein.
With small variations to this protocol, we can additionally determine if it is a transmembrane
protein, or a monolayer associated protein, but these won’t be explored in this animation.
There are many different types of membrane proteins, and here are just a few of them.
You’ll notice that some of them have exposure to the extracellular side, and some don’t.
In addition, there are proteins that have sugar groups added these are the things shown
in blue. When we have these sugar groups added, these proteins are called glycoproteins glyco
meaning sugar. In this scenario we have a cell and we have three different proteins
that were interested in determining their orientation. We’re interested in determining
if their extra cellular facing, and if they have carbohydrate groups making them glycoproteins.
So here’s the experimental setup, we have three different cell cultures for the same
types of cells that are grown. So each of these different cell cultures and different
compound is added. In one of the treatments a compound called FM is added, when this F
M reagent is added to the cells, any part of the protein with a portion on the extracellular
side will have FM attached, since it’s only able to access the outside of the intact cells.
In a separate set of cells, a compound called PAS was added. When the PAS reagent is added
to the cells it recognizes any protein that has a carbohydrate or sugar group on the outside
of cells. For the last treatment, nothing is added the serves of control to make sure
all our proteins are isolated properly in the following steps. Now that the cells were
treated with the appropriate markers, the PAS and FM markers, the membrane proteins
need to be isolated from the membrane environment. The way that this is done is by using detergents.
When mixed with membranes the hydrophobic ends of the detergents bind to the hydrophobic
regions of the membrane proteins. This then displaces the lipid molecules. Since the other
end of the different molecule is polar, the binding tends to bring the membrane proteins
into solution as a detergent protein complex. The result of adding detergents is that the
detergents replace the lipid bilayer and stabilize the hydrophobic membrane region of the protein.
In addition you get a byproducts a little bit little lipid packets as well. In this
scenario shown we’re showing the proteins with no treatment. However, the cells that
were labeled with FM or pas would retain the label in solution. So the FM would remain
attached to any piece of the protein that was originally on the outside of the cell,
and the pas treated cells the PAS would still be bound to the carbohydrate unites. Now that
the proteins have been isolated from the membrane, proteins in solution are heated and mixed
with SDS. This heat and SDS causes the protein to linearize, and is fully coated with a negative
charge that is equal to the mass of the protein. The protein mixtures are put in to wells in
a poly acrylamide gel matrix. Then they are run in an electric field. Because they have
been coated with a negative charge, they float towards the positive terminal. The bigger
proteins are slowed in the gel matrix compared to the small protein so they separate based
on size. To make the gel easier to work with, the proteins are transferred to a special
paper which then can be developed to see the different proteins. In this case we would
cut the gel into three pieces, and add a solution that recognizes the FM molecule, the PAS molecule
or binds to all proteins. The solutions were added to detect the markers and the bands
are developed .Now that you understand the protocol you should be able to analyze which
of these proteins here have an extracellular component and which are also glycoproteins.