electrophoresis relies on the principle of forcing charged molecules - it can be
DNA or protein - through a gel by electrical current. In the case of
protein separation the gel is polyacrylamide gel and it often prepared
with the detergent sodium dodecyl sulfate (SDS) so that you end up separating
the proteins based on the size alone and not the inherent charges of the
proteins. in contrast you may use native polyacrylamide gel separation and in this
case you separate based on size as well as mass to charge ratios.
so you first start off by extracting the protein from either tissue or cells. if
it's from tissue you have to first homogenise the issues then you go ahead
and once you have the individual cells you then apply a process that is... that
will allow you to extract proteins from the membrane, the cytoplasm or from the
nucleus and if you are interested in protein from just a particular compartment of
the cell then you use reagents and methodology that is optimized for
extracting proteins from that particular part of the cell. so once you
have your protein mixture this is the time that you're going to put it on the
gel and separate them. so most importantly, after the extraction
the final step before loading your sample onto the acrylic polyacrylamide
gel is the addition of SDS... of a buffer that contains sodium dodecyl sulfate. now
sodium dodecyl sulfate as we mentioned is an anionic detergent so
it makes it... it imparts an overall negative charge to each of the protein
in the sample and so you're able to separate based on size alone. gel
electrophoresis is really a process where you are forcing proteins through a
gel using electrical currents. following
electrophoresis of the proteins through the gel so that you have separation of the
proteins you want to transfer the proteins from the gel to a membrane. the
advantages of transferring to the membrane is that you are now able to do
lots of experiments with your proteins in a format that's more durable. so the
gel is fragile, it tears easily, falls apart easily but a membrane is much tougher and
much stronger. These membranes have very strong attraction - what is you termed
affinity - for proteins
flank the gels with filter paper and porous material that will allow the
proteins to move from the gel to the membrane as you apply electrical currents
the two membranes that are commonly used are nitrocellulose or polyvinyldine
difluoride (PVDF). when electricity is applied the proteins move from the poly
acrylamide gel onto the membrane where they bind tightly
following transfer of the proteins from the gel to the membrane you can check
whether you have sufficient transfer by staining the gel you want to use a
reversible stain because then you're going to be using the membrane for
probing with your antibody so you want to use a reversible stain and the most
common one is Ponceau S. now onto the blocking stage so as mentioned the
membranes that you use to a transfer the proteins have a very high affinity for
the proteins. so after the transfer you want to block any part of the membrane
that does not have protein bound to it - this is to prevent the
antibody that you will be subsequently using to detect your protein of interest,
from binding to the free surfaces that are available on the membrane and this
would... if that happens you're gonna get unspecific binding in addition to a
really high background. you can use milk skim milk as a blocking agent typically
we use five percent skim milk or you can use serum or even highly purified proteins
to block sites that are... that you didn't transfer proteins onto during the
transfer step. so you've got your proteins onto a membrane, you've blocked
the sites that did not have proteins bound -
to prevent a really high background signal, so then you're going to put the
primary antibody that will detect the antigens / the protein in the mixture that
you want to see if it's there, or you want to compare their levels. Before we
get to that let's talk about washing steps and the kind of buffer that you
need. so usually you can use phosphate buffered saline or Tris buffered saline
now phosphate buffered saline on tris buffered saline are generally
interchangeable, but if you're looking for phospho proteins so phosphorylated
proteins, then you want to stay away from phosphate buffer saline and use the
tris buffered saline. Also if you are using alkaline phosphatase conjugated
antibodies, as we will get into in a little bit, then you also want to stay
away from phosphate buffer saline and use your Tris buffered saline
so let's detect your antigen or protein of interest. so you want to use a primary
antibody that is designed for Western blotting. generally people... labs stay away
from primary antibodies that are directly detectable so they go for the
primary antibody, then they'll use a secondary antibody that is conjugated to
an enzyme such as alkaline phosphatase or maybe a fluorophore that you can then
detect... subsequently detect and that allows you to indirectly detect the
protein of interest via the primary antibody that you used. so your secondary
antibody is going to be whichever species the primary antibody was raised
in so if the primary antibody was raised in mice, then the secondary antibody will
be an anti Mouse antibody; if the primary antibody was raised in rabbit, then the
secondary antibody must be against rabbit antibodies etc
you dilute the antibody which will come as a stock - quite a concentrated stock -
and you dilute it in a wash buffer. so let's talk about what
you're going to use as a wash buffer then: so wash buffers typically is, as we
mentioned, the PBS or the TBS and you can spike in some some blocking
agent and this reduces unspecific binding or a high background, and you can
also put in a little bit... a tiny percentage of detergent and tween
tends to be a popular detergent. so we mentioned that the secondary antibody is
usually tagged or coupled with an enzyme or a fluorophore and in the case of the
enzyme when it... when there is a substrate present, it releases signal that you
detect if the protein you're looking for is in the mixture that you've
transferred or if it's the fluorophore, then the secondary antibody will allow
you to detect. so in the past secondary antibodies were coupled to radioisotope
so if you're in a lab that's still using that then that would be your detection
you would have instruments for detecting your
protein via radio isotopes. More and more antibodies that are
coupled to fluorophores are becoming really popular. so if your secondary
antibody is coupled to a fluorophore you will need specialized equipment for
detecting because you need an excitation light source to excite the fluorophore
in order to detect its light emission. the popularity of this method is also
because you can use more than one fluorophore and so you can multiplex. coupling
the secondary antibody to a fluorophore such as alexa flu or dye light fluorophore
are becoming popular because there is no substrate step that you need to
perform and so it's a lot faster. See you in the next video. All the best with your
western blot experiments!