Western Blot / Protein Immunoblot explained | YouTubeToText
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when working with proteins one cannot
get around the technique
called protein immunoblot or
simply the western blot western blotting
is a method to confirm the presence of a
specific target protein
in a sample before the analytical
technique western blood comes into play
everything starts with an sds page
upon treatment with sds all proteins in
a sample
are denatured and covered by negative
charges
the proteins have a similar master
charge ratio
and travel through the gel to the
positively charged anode
this allows to separate proteins
according to their molecular weight
after staining the polyacrylamide gel
protein bands become visible marker
proteins
as a reference help to determine the
molecular weight
of proteins in the sample this band here
for example
contains proteins with a molecular
weight of approximately
60 kilodalton however that will not
reveal
which proteins are present in this
sample it could be the protein of
interest which has a molecular weight in
this range
but the band could have also been caused
by
other proteins to confirm the presence
of a specific protein
a western blood has to be performed the
detection of the protein of interest is
based on
antibodies designed to exclusively bind
epitopes of
the target protein the western blot
setup starts with the gel containing
the separated proteins this is placed on
top of a membrane
often in nitrocellulose membrane
this membrane is capable to bind large
amounts of protein
later the idea is to transfer the
proteins
from the inside of the gel onto the
membrane
this gel membrane construct is flanked
on both sides
by filter papers all parts here are
soaked in buffer
to facilitate the electricity
transmission later
finally everything is fixed between the
cathode and the anode
the electric current pulls the
negatively charged proteins
which are located in the gel to the
positively charged a node
this transfers the proteins onto the
membrane
from now on the membrane has to be
incubated several times
the first step includes blocking
therefore the membrane is incubated with
milk
or bsa blocking should prevent
antibodies
from unspecifically binding to the
membrane or to other proteins
after blocking the membrane can be
incubated with the primary antibody
this antibody should be selected to only
bind to the protein of interest
after incubation with this primary
antibody a washing step will wash away
the unbound antibodies while not
removing the antibodies
that are bound to the protein of
interest the next step
involves the incubation with a secondary
antibody
which has to be specific for the primary
antibody
after washing only those secondary
antibodies remain
which are bound to the primary antibody
the detection of the protein
of interest is the last step in western
blood analysis
for that purpose the secondary
antibodies are conjugated to an
enzyme for many western blood assays
secondary antibodies are conjugated with
horseradish peroxidase
the protein of interest is bound by a
primary antibody
the secondary antibody binds to the
primary antibody
and when the membrane is incubated in a
substrate mixture
the enzyme converts the substrate
thereby producing a chemiluminescent
signal
that visualizes the protein band the
presence of the target protein in these
samples
has been confirmed make sure to
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