How To: Gel Electrophoresis and Western Blotting for Protein Analysis

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
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!

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