Western blotting technique | principle and step by step procedure
Summary
TLDRThis video tutorial delves into Western blotting, a technique for detecting proteins in a mixture. It covers the process from protein extraction to separation via SDS-PAGE, transfer to a membrane, and detection using specific antibodies. The method's sensitivity allows for the detection of nanograms of protein and is crucial for understanding cellular processes and protein interactions. The tutorial also explains the importance of blocking to reduce background noise and the use of primary and secondary antibodies for accurate protein identification.
Takeaways
- 🧬 Western blotting is a technique used to detect proteins in a mixture, different from Southern and Northern blotting which detect DNA and RNA respectively.
- 🔍 It is highly sensitive, capable of detecting nanograms of proteins even when mixed with other proteins.
- 🧴 The process begins with protein extraction from cells using detergent to break down the cell membrane, followed by centrifugation to separate the soluble protein components.
- 🔬 Separation of proteins is achieved through SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis), which separates proteins based on their mass and charge.
- 🚫 The gel is fragile, so proteins are transferred onto a more robust medium like nitrocellulose or nylon membrane for further analysis.
- 🖨 The transfer of proteins from the gel to the membrane is facilitated by applying an electric current, a process known as electroblotting.
- 🚫 To prevent non-specific binding of antibodies, a blocking step is introduced where the membrane is treated with agents like dry milk to cover non-target areas.
- 🔗 The primary antibody specifically binds to the target protein, but does not provide a signal for detection.
- 🔄 After the primary antibody binding, unbound antibodies are washed away, and a secondary antibody is applied which acts as a reporter.
- 💡 The secondary antibody is linked to an enzyme, often horseradish peroxidase (HRP), which upon reaction with a substrate, produces a detectable signal, such as light or color.
- 📝 The final signal, whether luminescent or colored, indicates the presence and location of the target protein on the membrane, allowing for its identification and analysis.
Q & A
What is Western blotting?
-Western blotting is a technique used to detect specific proteins in a sample. It involves separating proteins by size using SDS-PAGE and then transferring them onto a membrane to be detected with specific antibodies.
How does Western blotting differ from Southern and Northern blotting?
-While Southern blotting is used to detect DNA and Northern blotting for RNA, Western blotting is specifically used for detecting proteins. Southern and Northern blotting techniques are quite similar, but Western blotting involves different steps, including the use of antibodies for detection.
What is the sensitivity of Western blotting?
-Western blotting is a highly sensitive technique capable of detecting nanograms of protein molecules even in a mixture of other proteins.
What is the first step in the process of Western blotting?
-The first step in Western blotting is the extraction of proteins from the cell. This involves treating the cell with a detergent to break down the cell membrane, followed by centrifugation to separate the soluble protein components from cellular debris.
What is the purpose of SDS in Western blotting?
-SDS, or sodium dodecyl sulfate, is used in the electrophoresis step of Western blotting to denature proteins and give them a uniform negative charge, allowing them to be separated based on their mass during SDS-PAGE.
What is the role of the polyacrylamide gel in SDS-PAGE?
-The polyacrylamide gel in SDS-PAGE provides a matrix with different pores that allow proteins to be separated based on their size. Smaller proteins can migrate further through the gel, while larger proteins are hindered, resulting in bands of proteins based on their mass.
Why is it necessary to transfer proteins from the gel to a membrane in Western blotting?
-Transferring proteins to a membrane is necessary because the membrane is more robust and easier to handle than the gel. It allows for the application of specific antibodies for detection and is less prone to damage during the probing process.
What is the purpose of the blocking step in Western blotting?
-The blocking step is used to prevent non-specific binding of antibodies to the membrane. It involves applying a blocking agent, such as dry milk, to cover areas of the membrane where the target protein is not present, reducing background noise in the final detection.
What are primary and secondary antibodies in the context of Western blotting?
-Primary antibodies are specific to the target protein and bind directly to it. Secondary antibodies, which are anti-primary antibodies, bind to the primary antibodies and are often linked to an enzyme or a reporter molecule that aids in the detection of the target protein, such as through a colorimetric or luminescent reaction.
How is the presence of the target protein detected after the application of primary and secondary antibodies?
-After the application of primary and secondary antibodies, a substrate is added that reacts with the enzyme linked to the secondary antibody. This reaction produces a visible color change or light emission at the location of the target protein, indicating its presence on the membrane.
What is the significance of the enzyme HRP in Western blotting?
-HRP, or horseradish peroxidase, is an enzyme commonly linked to the secondary antibody in Western blotting. It catalyzes the conversion of a substrate, such as luminol, into a product that emits light, allowing for the detection of the target protein on the membrane.
Outlines
🔬 Introduction to Western Blotting Technique
The script introduces Western blotting as a technique for detecting proteins, distinguishing it from Southern and Northern blotting which are used for DNA and RNA detection, respectively. Western blotting is highlighted as a highly sensitive method capable of identifying nanograms of protein in a mixture. The script outlines the general process of blotting, emphasizing the three main stages: extraction, separation, and probing. It also mentions the use of Western blotting for detecting specific proteins, analyzing chemical reactions within cells, and understanding protein interactions.
🧬 Protein Extraction and Separation via SDS-PAGE
This paragraph delves into the first two stages of the Western blotting process: protein extraction and separation. Proteins are extracted from cells using detergents and centrifugation, resulting in a supernatant containing the protein mixture. The separation of proteins is achieved through SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis), where proteins are differentiated based on their size and charge. The process is described in detail, explaining how smaller proteins migrate further in the gel due to the presence of different pore sizes in the polyacrylamide matrix.
🖨️ Transferring Proteins to a Membrane for Analysis
The script explains the transfer process of proteins from the SDS-PAGE gel to a nitrocellulose or nylon membrane, a critical step before probing. This transfer is necessary because the gel is fragile and not suitable for further probing steps. The process, known as electroblotting, involves applying an electric current that moves the proteins from the gel to the membrane. The membrane acts as a stable and durable medium for subsequent analysis, allowing for the proteins' pattern to be preserved for further detection.
🔎 Probing and Detection of Target Proteins
The final part of the Western blotting process is described in this paragraph, focusing on the probing and detection of the target protein. After transferring the proteins to the membrane, a blocking step is performed to prevent non-specific binding of antibodies. The primary antibody, specific to the target protein, is applied and binds to its antigen. Following a washing step to remove unbound antibodies, a secondary antibody linked to an enzyme is applied. This secondary antibody acts as a reporter, binding to the primary antibody and enabling the detection of the target protein through a colorimetric or luminescent reaction, depending on the substrate used.
Mindmap
Keywords
💡Western Blotting
💡Blotting Techniques
💡Protein Extraction
💡SDS-PAGE
💡Electrophoresis
💡Transfer
💡Nitrocellulose Membrane
💡Blocking
💡Primary Antibody
💡Secondary Antibody
💡Horse Radish Peroxidase (HRP)
Highlights
Western blotting is a technique for detecting proteins, different from Southern and Northern blotting which detect DNA and RNA respectively.
Western blotting is highly sensitive, capable of detecting nanograms of proteins in a mixture.
The technique is used to identify specific proteins, understand chemical reactions within cells, and explore the total proteome.
Protein extraction involves treating cells with detergent to break down the cell membrane and release cytosolic components.
Centrifugation separates the supernatant containing proteins from the pellet containing cellular debris.
SDS-PAGE is used for protein separation based on size and charge, unlike DNA/RNA separation with agarose gel electrophoresis.
Proteins are negatively charged due to SDS treatment, migrating towards the positive end in the gel.
The process of transferring proteins from the gel to a membrane is called blotting, using an electric current.
Nitrocellulose or nylon membranes are used for protein transfer, as they are more robust than gels.
Blocking agents like dry milk are used to prevent non-specific antibody binding to the membrane.
Primary antibodies bind specifically to the target protein, but do not provide a detectable signal.
Secondary antibodies, linked to enzymes, act as reporters by binding to the primary antibodies.
Horseradish peroxidase (HRP) is a common enzyme used in conjunction with secondary antibodies for detection.
Luminal is a substrate that, when acted upon by HRP, produces light, indicating the presence of the target protein.
The detection of light can be captured on film, revealing the location of the target protein band.
Western blotting is crucial for identifying protein-protein interactions and their roles in cellular physiology.
The technique provides a visual confirmation of protein presence and can be used for further protein analysis.
Transcripts
[Music]
thank you
welcome back friends welcome to another
video lecture from Shamus biology and in
this video tutorial we'll be talking
about Western blotting we have been
talking about different types of
blotting techniques lately we've talked
about southern blotting Northern
blotting and this time we'll be talking
about the Western blotting blotting is a
technique of detecting macromolecules if
you are trying to detect DNA we use
solvent blotting if you want to detect
RNA we use Northern blotting and if you
want to detect proteins we use Western
blotting now northern blotting and
southern blotting has very very similar
techniques which uses mostly same type
of techniques and setup while Western
blotting is a little different compared
to them in Western blotting it is very
very sensitive reaction technique that
can even detect nanograms of protein
molecules in the mixture of other
proteins now there are different ways of
detecting a Target protein from a
mixture we can use chromatin
immunoprecipitation we can use like
coimino precipitation or antibody
mediated pull down assays but in case of
this Western blotting we detect very
minute amount of protein that is present
in a Cell fraction and a mixture and
Western blotting is a widely used
technique to not only detect a specific
protein but also to find out different
chemical reaction going on inside the
cell as well as to find out the total
proteome inside the cell as well as is
very important to understand about the
protein protein interactions for
different physiological cellular
purposes inside the cell so let us look
at the whole process
in the process of Western blotting again
in any blotting processes we rely on
three different stages the first step is
to extraction an identification of first
is the extraction of the molecule so in
this case the molecule is proteins so we
need to extract the protein content from
a cell what we do we simply treat the
cell with detergent that will break down
the cell membrane so membrane pore will
be formed and cytosolic components can
come out then we do the spinning of that
cell in the centrifuge that is going to
give us a supernatant and a pellet the
pellet contains all the cellular debris
like cell membrane organelles and all
the stuff while all the protein
components are suspended it's it's
soluble in the in the in the supernated
so take out the supernatant and we have
a protein mixture so start with that
point so we have a protein
mix
that is isolated from cell and the
protein mix contains so many varieties
of protein let's see this green one
let me draw some of the examples this
red one
that they have different concentrations
as well but they have different
varieties of
proteins in there okay so protein mix
then what we do the second step of any
blotting process is separation of those
mixture of molecules in this case we'll
do the separation of protein the process
of separation of protein that we use
is
electrophoresis
obviously
and the electrophoresis that we use to
separate protein molecules from each
other are known as
SDS page
sodium dodecil sulphate which is SDS
polyacrylamide gel electrophoresis known
as pitch so polyacrylamide jelly
electrophoresis is a process of
separating
all those protein mixtures from each
other
now in case of Southern and West
Northern blotting we have separated DNA
and RNA molecules ah with the help of
agarose gel electrophoresis but in case
of
in case of Western blotting we separate
proteins with page
it's a normal process of separating
protein molecules in this case the the
gel is made with polyacrylamide because
acrylamide can attach with each other to
make a cross linking and form a polymer
so they are separated based on forming
this polymers there
okay so they are separate based on that
so once they separate so so let me draw
this whole thing of SDS page
in one way we put all those known
proteins now SDS page is a type of
electrophoresis process which helps
separating proteins based on their
charge
size
and Mass
mainly by their size and mainly based on
their mass and and also shape ah mostly
not size you can say the shape
so its mostly the structural feature
that helps separating proteins based on
that charge the separation of protein
based on the charge requires some some
more modifications in the SDS page but
mostly this separated by their mass now
the idea is as this this is a
polyacrylamide gel it carries so many
different pores in between
if the protein is smaller then that
protein can migrate further but if a
protein is large that protein will not
migrate much that protein will halt at a
specific point so the smaller proteins
will migrate further so we'll get bands
in in a in further directions while the
larger proteins
will be lacked behind during the
migration and let us say the migration
direction is this OK and again the
directionality of this migration that we
are talking about is also towards the
positive end
because the proteins that we are talking
about here they are negatively charged
because they treat it with SDS so the
negatively charged so they will migrate
towards the positive end
okay so that is the idea
of SDS page so with the help of this SDS
page we separate the mixture of proteins
based on their Mass
and charge means or not let's talk about
charge here we just talk about the mass
and separation so now let's say we
separated all the mixture of proteins
based on their mass and then what we do
we need to use a probe to find out our
Target protein right
that is the idea this is the second step
of this blotting process every blotting
process consists of three state the
first step is extraction and isolation
second step is the electrophoresis third
step is a probing
and before and between the
electrophoresis and probene a new unique
process to be done that is the transfer
of the content that is present in the
gel onto a membrane paper why it's
required because you know we want to add
probe and that probe can bind to
different regions of this gel it should
be properly attached to the Target
protein only
right and gel is really really fragile
because the content of the gel is not
that strong so we cannot do rest of the
process of probing and all this
reactions using using this gel
so we need to transfer the components of
the gel exactly like the same imprint
into a nitrocellulose membrane or nylon
membrane filter
for this process of transfer that is the
reason we call it as a western blotting
that's the term blood comes in because
it's simply blotted and simply taking
the imprint of the gel replica onto a
natural cellulose membrane so you take
the nitrocellulose membrane and we put
on top of this gel and then we apply
current that will help flowing this
protein molecules from the gel towards
the membrane and those proteins will be
attached to the membrane because this
membrane is kind of a like sticky
towards those macromolecules okay so
let's see how this process is exactly
done so let us assume SDS page is
conducted now if you don't have any idea
about SDS page and if you don't know
about the details of SDS page I will
recommend you to watch my video on SDs
page there are plenty videos three or
four videos available in my channel
regarding SDS page you can watch that
anytime but for now let us say the SDS
page is done and we have the gel ready
then how we transfer this component the
way to transfer this component in case
of Western blotting
Electro illusion that is the process of
transferring the component from the
sdsgel to the Nitro cellulose membrane
so what we do let us assume this gel as
a as a side view so if you look at this
gel as a side view it will look
something like this this is the gel
we put it in horizontal line
and what we do now we also put
the nitrocellulose membrane let us say
this is the Nitro cellulose membrane
this is the membrane
and this is the gel
OK and then what we do is we apply
electrodes in both these directions okay
so we will apply
positively charged electrodes towards
the membrane negatively charged
electrodes on this towards the gel side
that is the idea that we have
ok now what we do once we put the
electrodes the current will flow
from this negative towards the positive
electrode and as the current is Flowing
from negative to positive the protein
molecules that are present
here because you know they this this
membrane is in direct contact with the
gel so let me do that
it is in direct immediate contact with
the gel
it's an immediate contact so what is
going on once we apply the current the
protein molecules start flowing
from the gel
to the nylon filter
that is the process and we also put
everything in the buffer definitely we
need to put them in buffer and
electrolytes so that they can flow so
once this process is done what is the
importance of this process plotting that
is the transfer of those components and
proteins from the SDS page to the
nitrocellus membrane because the
membrane is much easier to handle it's
more strong and we can we can use this
membrane for other purposes so once this
blotting is done once the transfer is
complete then we take the membrane
then we take this membrane here let me
take the membrane let's draw the
membrane
this is the membrane and let us say in
this membrane we find out ah some region
of proteins different regions or bands
that we can see for example this
this
is for example
everything is present in the membrane
now the third important stage that is a
probing
and hybridization in case of DNA and RNA
blotting we simply use complementary
strands for the hybridization assay but
in this case hybridization won't work
because it's protein so we need to use
any sort of detector
that can go and bind to the specific
Target protein of our interest for that
you know
we have antibody
antibodies can be specific it can attach
to specific proteins so it design
antibody against our Target protein and
we apply that antibody to go and
interact with our Target protein among
the rest of the proteins in the
nitrocellulose membrane
but before doing that we do another step
which is called as a blocking blocking
is a step where we apply some blocking
agent example one of one such blocking
is Agent is dry milk we apply dry milk
so that that portion covers most of the
area because that will not bind to the
protein or targeted protein areas but
mostly that will bind to the rest of the
Nitro cellulose membrane why it is
important because if we don't do the
blocking State and simply put the
antibody antibody are specified antibody
still can bind with some unspecific
reaction with different regions antibody
should bind with the protein but along
with that it might also bind with some
extra other regions of the of the
membrane that will give us some noises
background noises because it will be
giving us a positive false ah positive
signal or its a kind of a background
noise so to reduce this noise we won't
allow this antibody to be interacting
with the rest of the member membranes
region so that is the region we we kind
of covered the rest of the membrane with
all this blocking edges so with the help
of buffer and blocking agents we simply
block the rest of the portion of the
membrane and we also use some mild
detergent as well so it's covering rest
of the blocking area then we apply the
primary antibody and the antibody is
very specific and so much specific
towards the target protein it will not
allow to any blocking to prevent it so
it is going to bind for example say this
is the target this is the target
protein so antibody can bind to the
Target protein and even multiple
antibodies can bind to one protein
content
OK that is the first step and the
antibody that we applied for the first
time is known as the primary antibody is
known as
primary
antibody because this is the first type
of antibodies that we apply which will
directly bind
it will directly bind to the Target
protein but primary antibody lacks in
specific part that it will not show us
the result because it's not a reporter
it can only directly bind to the Target
but it will not give us any fluorescence
or any light or anything of that
it's just for the proper binding so
after binding of the primary antibody we
we do the process of washing out so the
rest of the Unbound antibodies are
washed away
then we also add a secondary antibody
secondary antibody and now this one is
important because secondary antibody is
acting as a reporter
because this secondary antibody is
anti-primary antibody that means if this
is the primary antibody
one degree means primary
secondary antibody will be anti-primary
that means secondary antibody have the
capability to bind with the primary
antibody FC portions
that will be the interaction between
primary antibody and secondary will be
targeted against primary so it will
attach to the primary now secondary is
attached with any sort of enzyme
that is why it is called as a reporter
primary goes and bind to our Target
protein let us say this is the target
protein the green color let us say this
is the target protein primary binds to
the Target protein we applied we do the
washing step then you apply secondary
antibody secondary antibody is attached
to the enzyme it is binding to the
primary antibody so we have a proper
binding done
now here the secondary antibody attached
to the enzyme so if after this we add
the substrate
and the substrate will be converted into
product by the attached enzyme that is
present there with the secondary
antibody FC region it will be converting
the substrate into product
now the product that we prepare
sometimes give us color so if we do this
reaction
in in different big in different let's
say trays and stuff we get some color
generates in a specific region of this
whole membrane that is going to tell us
that that is the region where the target
proteins present
or sometimes the this this enzyme or the
chemical molecule whatever present there
upon giving a specific substrate it will
convert it into a product and it will
generate light as a byproduct
and that is very common in case of the
western blotting technique and detection
and the enzyme that we use is known as a
horse release peroxidase hrp horse
reduce peroxidase once you use hrp
remember hrp here is attached
and then we add the luminal
it converts the luminal and break it
down and as a byproduct of this reaction
it provides us light
and we can show the light that is coming
out and that can be easily detected with
a extra film because that that film is
completely uh like dark and the dark you
will see those gray faded bands due to
the generation of the light that's going
to give us the idea about where exactly
the target protein is present in that
mixture of SDS page in the in the
picture of the SDS page so let's say you
want to find out that protein and cut it
out you can still do that because if you
see where exactly the light is coming
from so when you develop the film you'll
only see only one band
and in this case let's say the band is
kind of here
so then you can simply look at this this
SDS page
and you find out the band in the page so
if you want to take that protein out you
can simply go there and cut that portion
out and do the rest of the job
so that is the process
you see
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