hello in this little video lecture I'm
going to talk briefly about Western
blotting since that's something we'll be
doing this week in the lab Western
blotting first of all what is it as you
see there written on the screen it's an
analytical technique that's used to
detect very specific proteins in your
sample based on two distinguishing
properties of the proteins first is
molecular weight and the second is
antibody binding specificity so the
proteins need to first be separated
based on molecular weight which we know
we can do pretty easily now with an SDS
page running a gel to separate them
based on protein and then what we do
next in Western blotting is we will blot
those proteins from the gel onto a
membrane and then probe the membrane
with antibodies that are specific for
the protein of interest a little diagram
here on the side shows you the gel
blotting and then the probing with the
antibodies not too difficult sample prep
we've done this before depending on the
cells and the or the tissue or where the
source of the proteins sample prep could
be incubating with lysozyme and grinding
it up all sorts of different ways and
then the gel electrophoresis which we
know the SDS page allows us to separate
those proteins based on size shows you a
picture there of a typical SDS page gel
that has been stained we will probably
not stain ours first since we're going
to be probing with antibodies there's no
need to stain to be able to see the
proteins first transfer here's something
here's the new part that we have not
done that that you will be doing this
week in the lab the transfer is going to
use the same box that we used for the
SDS page to do the transfer now once the
proteins have been separated vertically
in the gel we're then going to transfer
them from the gel onto a membrane so
we're going to have to set up this this
fancy sort of sandwich as you can see
here are showing on the right side the
layers of the sandwich we're going to
start with a the gel holder the black
side will be down that would be towards
the cathode so we know the proteins are
negatively charged so they are going to
move in this direction
from the gel onto the membrane so
there's this sandwich there's some fiber
pads as you can see in the top of the
bottom there's also filter paper that
will help in the transfer keeping the
charge even by keeping the whole sample
wet there's also then the gel in the
middle right here and the membrane which
will lay right on top of the gel and we
will have the transfer of the proteins
once we squish that all together stick
it in the the electrophoresis chamber
and then we run the current actually
going across the gel this way as opposed
to going up and down that we've seen
before before we talk about the probing
we have to talk a little bit about
antibody basics probably familiar with
antibodies you know they're produced in
response to presence of a certain
antigen and they are highly specific
there are some things that are that are
similar about all antibodies you can see
here on this this diagram you have
there's two heavy chains in a typical
antibody there's one and then here's the
other and then there are two light
chains light simply because they're
shorter and there's a constant region
that's what the C stands for and then
there's also variable regions on the
ends here to get that typical Y shape
but there's two long heavy chains held
together by cysteine cross-bridges
the light chain and heavy chain also
held together by cysteine cross bridges
and then at the end here you have an
area which is where the antigen will
bind so you see that and there is the
antigen binding site and reach anybody
there are two of them one on each side
here where the it will specifically
stick to a particular antigen based on
its shape these oome in on that and look
on the right side here you can see the
specific antigen binding to that antigen
binding site the actual part of the
protein that sticks in that antigen
binding site is known as the epitope is
you can imagine a protein that is very
large has number of different sort of
pieces or structures sticking off for so
you could have antibodies that are
specific to one epitope or one area of
the protein and then a different
antibody that's specific to another area
of the protein or a different epitopes
so you can have multiple antibodies that
will recognize the same antigen so those
that's the quick and dirty version of
antibody so now we can talk about the
next step in the Western blotting once
we've
onto the membrane the first thing we
will need to do is block the membrane
noise we want to cover every other area
the membrane with a neutral protein
we're going to use casein or milk
protein so that any antibodies that once
we add the antibodies they won't be able
to stick to the membrane since the
membrane is sticky to proteins and
antibodies our proteins that anybody's
could stick on the membrane and places
we don't want them to so the first step
will be to block the membrane now is
cover up all the areas where there are
protein bands with casein after we do
that in Part A here we will add the
primary antibody the primary antibody is
specific to the protein we're after it
will bind specifically to the bands that
were on the SDS page gel in the exact
same locations that they've migrated so
this first
supply area here shows you the primary
antibody stuck to the antigen that is
actually on the nitrocellulose membrane
once we've bound the primary anybody
we'll wash off the extra put on a
secondary antibody which will be
specific to the primary antibody and
also have conjugated to it
a reporter enzyme in this case we're
going to use horseradish peroxidase
that's with the HRP in the HRP
conjugated antibody down here our step
represents that secondary antibody
recognizes the primary antibody as its
antigen so if anywhere there is primary
antibody bound the secondary anybody
will also bind and then once we wash off
the extra secondary antibody we will
have bound to the antigen a primary
antibody and a secondary antibody with a
reporter enzyme we then add the color
detection reagent which in this case is
the substrate for the horseradish
peroxidase and it what it will do is
produce a colored product which will
stick to the membrane so anywhere where
there's a primary antibody secondary
antibody and then the substrate we will
get a band very similar to what it
looked like on the gel so that's the
quickie for the blocking and detection
it was
walking to cover the membrane and all
the spots that there's no protein
primary antibody to stick to our target
for our targeted protein secondary
anybody which will stick to the primary
anybody and then the development stage
and that is it