Western blotting technique | principle and step by step procedure

Summary

This 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

00:00

🔬 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.

05:02

🧬 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.

10:03

🖨️ 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.

15:04

🔎 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.

Keywords

💡Western Blotting

Western blotting is a laboratory technique used to detect specific proteins in a sample of tissue homogenate or extract. It is one of the core vocabulary in the script as it is the main topic of the video. The process is described as being highly sensitive, capable of detecting nanograms of protein in a mixture, and is essential for identifying protein-protein interactions and the total proteome within a cell. The script outlines the entire process, from protein extraction to the final detection using antibodies.

💡Blotting Techniques

Blotting techniques refer to a group of methods used to transfer and detect specific molecules, such as DNA, RNA, or proteins, onto a membrane. In the script, different blotting techniques are mentioned, including Southern blotting for DNA and Northern blotting for RNA, with Western blotting being the focus. These techniques are fundamental to molecular biology and genomics research.

💡Protein Extraction

Protein extraction is the initial step in Western blotting where proteins are isolated from cells. The script describes treating cells with detergent to break down the cell membrane, followed by centrifugation to separate the soluble protein components from the cellular debris. This step is crucial as it provides the protein mixture that will be further processed in the blotting procedure.

💡SDS-PAGE

SDS-PAGE, or Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, is a method described in the script for separating proteins based on their size. It is a critical step in Western blotting that allows for the visualization of proteins as distinct bands on a gel, facilitating the identification and analysis of specific proteins within a complex mixture.

💡Electrophoresis

Electrophoresis is the process used in blotting techniques to separate molecules, such as proteins, based on their charge and size. In the context of the script, electrophoresis is specifically applied to proteins in the form of SDS-PAGE, which is essential for preparing the proteins for transfer onto a membrane in Western blotting.

💡Transfer

In the script, transfer refers to the process of moving the separated protein bands from the SDS-PAGE gel onto a membrane, such as nitrocellulose or nylon. This step is unique to Western blotting and is necessary because the membrane is more stable and suitable for subsequent probing steps to detect the target protein.

💡Nitrocellulose Membrane

A nitrocellulose membrane is a type of porous membrane used in Western blotting to which proteins are transferred from the gel. The script mentions that this membrane has an affinity for proteins, allowing them to adhere to its surface after the transfer process, making it an ideal support for the detection of specific proteins.

💡Blocking

Blocking, as described in the script, is a step in Western blotting where a blocking agent, such as dry milk, is applied to the membrane to prevent non-specific binding of antibodies to the membrane. This is important to reduce background noise and ensure that the antibodies only bind to the target protein, thereby increasing the specificity of the detection.

💡Primary Antibody

The primary antibody is a specific type of antibody designed to bind to the target protein of interest. In the script, it is mentioned as the first antibody applied during the probing step of Western blotting. It is essential for the process as it directly interacts with the target protein, setting the stage for detection.

💡Secondary Antibody

The secondary antibody, as discussed in the script, is an antibody that binds to the primary antibody and is typically conjugated with an enzyme or a fluorescent tag. It acts as a reporter in Western blotting, amplifying the signal from the primary antibody's binding event, making the detection of the target protein more visible and quantifiable.

💡Horse Radish Peroxidase (HRP)

Horse Radish Peroxidase (HRP) is an enzyme used in the script's description of Western blotting as part of the detection process. When the secondary antibody is conjugated with HRP, it can catalyze a reaction with a substrate, such as luminol, to produce light. This light emission is a visual indication of the presence of the target protein, allowing for the detection and analysis of specific proteins on the membrane.

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