Another method [that] we can use in biochemistry to help us locate a specific
Protein that we wanted to study is western blotting and just like ELIZA methods western blotting also utilizes
antibodies
So the entire point in a western blot is to form a specific antibody that can bind [onto] [that]
Protein that we want to locate and when this process of binding takes place it somehow allows us to visualize
Where that protein is as we'll see in [just] a moment
So let's begin by supposing we have a sample of proteins now
Where did the sample actually come from so suppose? We have a collection of cells and inside the cells we have
proteins and the protein that we want to actually locate it's down within the cells
So the way that we're going to isolate the proteins from inside the cells is by
fractionating our cells and then placing that fraction into a set into a
differential centrifuge machine, and then as a result of centrifugation, we're going to basically
Isolate the proteins found inside our cell now once we isolate the protein sample
We take the sample of proteins and place it into a gel electrophoresis setup. So we use
SDS polyacrylamide gel electrophoresis
and that allows us to use an electric field to separate the proteins based on their size, so
This is what we get following our gel electrophoresis
Experiment, and so this band
Represents those proteins that are the largest in size and this band represents the proteins that are the smallest in size
Remember in SDS Polyacrylamide gel electrophoresis
these proteins move as a result of charge as a result of being inside an electric
Field and the largest proteins move the slowest because they have the greatest resistance
Now once we form the set up we also basically want to form the monoclonal
Antibody that is specific for [that] protein that we want to study and that protein that we want study
We call [the] antigen so we synthesize this monoclonal
Antibody we call the primary antibody that can bind
To that antigen the protein that we actually want to locate now once we form the monoclonal
Antibody and once we run gel electrophoresis we then transfer that
Separate protein sample onto a polymer sheet and this step is basically done to make it easier [for] us to work with our
separated protein mixture
Now the next step is to is [to] take this polymer sheet
And we mix it with this antibody that we formed in step one and once we mix these two mixtures this
Antibody the primary antibody it will basically bind [onto] the proteins found in one of these
Bands because one of these bands will contain that antigen that is specific for this protein and so let's suppose
It's the second band from the top that
Contains the proteins that we want to study and so this antigen or this
Antibody will bind to the antigen the protein found in the second bin
so the monoclonal antibody is added onto the polymer sheet containing the
Separated protein mixture and it only binds on to the antigen onto the protein that we actually are interested in
Now in step number five we synthesize yet another antibody
And now we synthesize an antibody that is
radioactively-labeled
And what that allows us to do in the final step is to visualize exactly where that protein is now
The antibody that we want to synthesize the second time around [this] antibody and step number five is known as the secondary
Antibody and the Secondary antibody is made so that it binds [onto] that
Primary antibody that we formed in step one so once we form this secondary
Radioactively labeled antibody, we then mix this mixture of antibody with our
limor sheet that contains
That antigen that is bound to the primary
Antibody and what happens is [because] this was formed to bind [unto] this blue primary antibody this
Complex is formed so the polymer sheet containing the antibody antigen
complex is now exposed to a solution containing the
radioactively labeled antibody
So we take the green mixture of antibodies we reacted with the polymer sheet that contains
the blue Antibody bound [onto] the protein found on this second band now the
radioactive antibody will only bind to this band and now the other bands because only
This second band contains this protein the antibody bound unto the antigen because this was built
Specifically to bind on to this primary blue
Antibody now in the final step we basically take an x-Ray film we expose it to
X-Rays and what that allows us to do is allows us to visualize
exactly where this
radioactively labeled antibody has bound to and because it is bound to the protein found on the
Second band and none of these other bands contained this one bound to them that means
[only] the second band will appear dark on an X-Ray film and that allows us to exactly pinpoint
Where that protein is the protein [that] we want to study and now we can take the protein?
isolate the protein from this particular location on that X-Ray film