Eastern blotting

Summary

This video tutorial offers a concise introduction to various blotting techniques in molecular biology, focusing on Southern, Northern, and Western blotting. It explains how these methods are used to identify specific DNA, RNA, and protein sequences. The video also delves into Eastern blotting, a technique for detecting post-translational modifications in proteins, which is crucial for understanding protein function and localization. The tutorial clarifies the differences between blotting techniques and highlights the importance of antibodies in targeting modified regions of proteins.

Takeaways

• 🧬 **Blotting Techniques Overview**: The video introduces various blotting techniques used in molecular biology, including Southern, Northern, Western, and Eastern blotting.
• 🔍 **Southern Blotting**: This technique is used for identifying a specific segment of DNA sequence from a gel after electrophoresis.
• 📄 **Transferring DNA**: DNA from the gel is transferred to a nitrocellulose paper, which is easier to handle and binds with DNA.
• 🔬 **Probes for Detection**: Probes, either radioactive or fluorescent, are used to bind to the specific DNA region of interest for detection.
• 📚 **Northern Blotting**: Similar to Southern blotting but used for RNA, this technique was developed in 1975.
• 🏥 **Western Blotting**: Focused on proteins, this method was developed in 1979 and 1981 to identify specific proteins from a gel.
• 🌏 **Eastern Blotting**: Introduced later, this technique is used to detect post-translational modifications (PTMs) in proteins.
• 🔄 **Post-Translational Modifications**: PTMs like glycosylation, methylation, and acetylation are crucial for determining the function and location of proteins within a cell.
• 🏷️ **Addressing Proteins**: PTMs act as 'addresses' for proteins, directing them to specific cellular locations, such as mitochondria or outside the cell.
• 🛠️ **Eastern Blotting Process**: Involves transferring proteins from an SDS-PAGE gel to a membrane, then using antibodies to detect modified regions of proteins.

Q & A

What is the primary purpose of blotting techniques in molecular biology?

Blotting techniques are used to identify and detect specific segments of DNA, RNA, or proteins from a gel after electrophoresis.

What are the three main types of blotting techniques mentioned in the script?

The three main types of blotting techniques mentioned are Southern blotting, Northern blotting, and Western blotting.

What does Southern blotting involve and why is it called so?

Southern blotting involves the identification of a specific segment of DNA sequence from a gel. It is named after the scientist who developed it, E.M. Southern.

How is Northern blotting different from Southern blotting?

Northern blotting is used for RNA instead of DNA, and it was named as such to reflect the directionality after Southern blotting.

What is the main focus of Western blotting?

Western blotting is used to detect specific proteins from a gel, and it involves the use of antibodies to bind to the protein of interest.

What is Eastern blotting and when was it developed?

Eastern blotting is a technique used to detect post-translational modifications in proteins. It was formulated in 2009 and began to gain attention from then on.

What are the two types of Eastern blotting mentioned in the script?

The two types of Eastern blotting mentioned are Middle Eastern and Far Eastern blotting.

Why is the production of specific antibodies crucial in Eastern blotting?

In Eastern blotting, the production of specific antibodies is crucial because they must target the modified regions of proteins, not just any region, to detect post-translational modifications.

How do primary and secondary antibodies differ in the context of blotting techniques?

Primary antibodies bind directly to the protein of interest, while secondary antibodies bind to the primary antibody and often carry a fluorescent tag for detection.

What is the significance of post-translational modifications in proteins?

Post-translational modifications are significant because they can alter protein function, stability, and cellular location, acting as 'addresses' for where the protein should be delivered within or outside the cell.

What is the role of GGI bodies in the context of protein modifications mentioned in the script?

GGI bodies (Golgi apparatus) play a role in post-translational modifications of proteins, where proteins are modified and packaged into vesicles for their final destination within or outside the cell.

Outlines

00:00

🧬 Introduction to Blotting Techniques

This paragraph introduces the viewer to various blotting techniques in molecular biology and recombinant DNA technology. The speaker explains that blotting is a method used to identify specific segments of DNA or RNA from a gel. Southern blotting is highlighted as a technique for identifying a specific DNA sequence, while Northern blotting is used for RNA. The process involves transferring DNA from a gel to a nitrocellulose paper, which is easier to handle, and then using probes to bind to the DNA of interest. The probes can be radioactive or fluorescent, depending on the budget. The paragraph also touches on the development of Eastern blotting, which is used to detect post-translational modifications (PTMs) in proteins, such as glycosylation, methylation, and acetylation. These modifications are crucial for determining the final destination of proteins within or outside the cell.

05:02

🔬 Eastern Blotting: Detecting Protein Modifications

The second paragraph delves into the specifics of Eastern blotting, a technique developed to detect post-translational modifications in proteins. The process is similar to Western blotting, starting with SDS-PAGE to separate proteins. After transferring the proteins onto a nitrocellulose paper, antibodies are used to target specific modified regions of the proteins. The speaker emphasizes the importance of having antibodies that bind only to the modified sections, distinguishing Eastern blotting from Western blotting, which targets specific amino acid sequences. The paragraph also mentions the types of antibodies used, such as primary and secondary antibodies, with the latter often carrying a fluorescent tag for visualization. The speaker suggests watching a separate video for a deeper understanding of primary and secondary antibodies. The paragraph concludes by encouraging viewers to like, subscribe, and share the video for more educational content.

Keywords

💡Eastern blotting

Eastern blotting is a variation of Far Western blotting used to detect post-translational modifications (PTMs) in proteins. It is a significant method in molecular biology to understand how proteins are modified after translation, which can affect their function, location, and interactions within the cell. In the video, Eastern blotting is introduced as a technique that targets modified regions of proteins, distinguishing it from Western blotting which targets specific amino acid sequences.

💡Western blotting

Western blotting is a widely used technique in molecular biology for detecting specific proteins in a sample. It involves separating proteins by size using gel electrophoresis, transferring them to a membrane, and then probing with antibodies to visualize the protein of interest. The video explains that Western blotting is foundational to understanding how proteins are identified and quantified, and it serves as a comparison to Eastern blotting.

💡Post-translational modifications (PTMs)

Post-translational modifications are chemical alterations that occur to proteins after their synthesis. These modifications, such as phosphorylation, glycosylation, and acetylation, can significantly affect protein function, stability, and interaction with other molecules. The video emphasizes the importance of PTMs in directing proteins to their correct cellular locations and how Eastern blotting is used to detect these modifications.

💡Primary antibody

A primary antibody is a specific type of antibody that binds directly to the target antigen, in this case, a modified region of a protein. The video script mentions that for Eastern blotting, primary antibodies are crucial as they target the modified regions of proteins, which is a key difference from Western blotting where they target specific amino acid sequences.

💡Secondary antibody

A secondary antibody is used in conjunction with a primary antibody to enhance the detection signal in immunoassays like blotting. It binds to the primary antibody and is often conjugated to a fluorescent tag or an enzyme that can produce a visible signal. The video explains that in Eastern blotting, a secondary antibody may be necessary if the primary antibody does not carry a fluorescent tag.

💡SDS-PAGE

SDS-PAGE, or sodium dodecyl sulfate polyacrylamide gel electrophoresis, is a technique used to separate proteins based on their size. The video script describes how proteins are first separated using SDS-PAGE before being transferred to a membrane for blotting, which is a standard procedure in both Western and Eastern blotting.

💡Nitr cellulose paper

Nitr cellulose paper is a type of membrane used in blotting techniques to transfer proteins or DNA from a gel to the paper for further analysis. The video mentions that this paper is used because proteins and DNA can bind to it, making it easier to handle than the gel itself.

💡Fluorescent probes

Fluorescent probes are molecules that emit light when excited, often used in molecular biology to visualize and detect specific molecules. The video script contrasts fluorescent probes with radioactive probes, noting that while fluorescent probes are more costly, they provide a way to visualize the presence of DNA, RNA, or proteins during blotting.

💡Radioactive probes

Radioactive probes are isotopically labeled molecules that can be detected by their radioactivity, used in various biological assays including blotting techniques. The video mentions that radioactive probes are a cost-effective alternative to fluorescent probes, allowing for the detection of DNA, RNA, or proteins by developing a radiogram.

💡Gel electrophoresis

Gel electrophoresis is a method used to separate molecules, such as DNA, RNA, or proteins, based on their size or charge. The video script describes how DNA is separated by gel electrophoresis before being transferred to nitrocellulose paper in Southern blotting, highlighting the importance of this technique in molecular biology.

Highlights

Introduction to Eastern blotting and its significance in molecular biology.

Explanation of different blotting techniques: Southern, Northern, and Western blotting.

Southern blotting for identifying specific DNA sequences.

Northern blotting for RNA detection, developed after Southern blotting.

Western blotting for protein identification, developed in 1979 and 1981.

Eastern blotting for detecting post-translational modifications (PTMs) in proteins.

Importance of PTMs in determining protein function and localization.

The process of Eastern blotting, including SDS-PAGE and transfer to nitrocellulose paper.

Use of antibodies in Eastern blotting to target modified regions of proteins.

Difference between primary and secondary antibodies in blotting techniques.

Challenges in producing antibodies specific to modified protein regions.

The role of the F(ab)' portion of antibodies in binding to specific protein regions.

The key difference between Eastern and Western blotting in targeting modified vs. unmodified protein regions.

The practical application of Eastern blotting in molecular biology research.

Encouragement to subscribe for more educational content on molecular biology.

Transcripts

hello guys in this video tutorial very

quickly I'll be talking about Eastern

blotting you probably heard that name

before and confused about what is it

there are many different types of

blotting techniques that are available

in molecular biology and recombinant DNA

technology lab uh it is mainly the ma

important things are uh the southern

blotting the northern blotting and

western blotting so this is a very basic

uh slide to demonstrate you different

types of blottings that are available

most popular ones Southern blotting and

Northern blotting because Southern

blotting means in all this kind blotting

means the identification of a DNA

specific segment of a DNA sequence uh

from from the gel that we have because

once we are doing we are separating DNA

based on their lens using the

electroforesis process G electrophoresis

technique after the gel electroforesis

we get a gel where we have bands of DNA

based on their length uh in KB or

kilobase pair we take that gel out we

transfer that gel into a nitr cellulous

paper uh because the paper is easy to

handle gel is fragile it can break and

it's very very fragile most of the time

so we take that uh that uh vile I mean

take that natural cellulose paper with

the DNA because DNA will bind with the

natural cellulous paper and then we uh

what we do we just make that DNA single

standard first and then we add some

probe that can specifically bind to the

region of the DNA we want to find and

that is called the blotting we use

certain type of probes like we can use

radioactive probes or we can also use

floresent probes it depends on us what

amount of budget we want to spend

because fluent probes are costlier than

radioactive probes so after the process

is done probing is done then we rest of

the part will be looted and if it's a

radioactive probe we develop a radiogram

where we see the presence of our desired

DNA or na or frin uh but in case of uh

in case of fluoresence probe also the

fluoresence coloration will visualize

the presence of the DNA RNA or protein

so if you do this blotting technique for

DNA it is called Southern blotting

actually it's developed by uh the

scientist Southern that's why according

to his name it is it's nothing to do

with the direction but once it is

developed uh people try that why it's

developed for DNA so we can do that for

RNA also right so try it for RNA it

works and they call it that first one is

Southern so let's make it Northern it's

like a directionality then they go for

Northern blotting for RNA is discovered

again in 1975 itself U Northern blotting

is developed after that Western blotting

is developed in 1979 and 1981 Western

blotting is for proteins the same thing

separation and finding a specific

protein from a blood and gel that is

called the Western blood these are the

three major uh very important blotting

techniques that are available but also

now we have Eastern blotting to find out

this is actually mainly a a version of

Far Western blotting mainly uh side

version Eastern blotting means uh this

is a blotting process for proteins but

to find out ptms post transational

modifications because you know in ukar

there are multiple post transational

modifications are possible Right like

glycosilation methylation acetylation

sumolation so these are the

modifications that are done that are

being done inside those what we

say you

[Music]

know GGI

bodies are the GGI bodies those

modification will be done because ER is

the place where all the proteins are

made from this ER those proteins are

taken inside the GGI where it is

modified and finally GGI is a chamber

which will put the proteins in vesicles

for their Final Destination it could be

the delivery of the protein outside the

cell those will be secretary proteins

like enzymes and minum hormones and many

things in some cases it can be uh

protein to be delivered into a specific

location of Cell itself like inside

mitochondria or inside plased in case of

plants so those post transational

modifications play a vital role for

designating where exactly the protein

will go just like it's if you say this

is a postal system GGI body is a post

office and uh those letters are vesicles

in that case post transation

modification act as the address where

exactly that protein will go right so

the post transation and modification

will act as the address so it's very

very important so we need to detect some

kind of post transation modification

inside the cell because a specific type

of post transation modification carries

a specific message a specific

modification can tell a protein to be

delivered to mitochondria on other hand

a different modification can tell that

protein to be delivered outside the cell

uh the secretary pathway so that's why

the post translation modification

determination is another important

challenge for us and that's why it's

developed it's developed early I mean

very late in 1982 it's formulated but

after that is in 2009 from 2009 Eastern

blotting slowly start to take over and

there are now two types of Eastern

blotting Middle Eastern and Far Eastern

we don't need to go through that part

but just know basics of Eastern blotting

to detect the post transation

modifications in proteins how could you

detect that again the process of

blotting will be the same because

obviously like other proteins Movement

we have SDS page remember because we are

talking about proteins so we have sodium

doicy sulfate polyacryamide gel

electroforesis SDS page so after this

SDS page is done we have the we have the

SDS page gel in our hand and then we can

transfer this SDS page protein to our uh

filter I mean the paper nitr cellulous

paper in this case we use different

kinds of paper

uh available in the market readily so

you transfer it into the paper once it's

transferred to the paper then the rest

of the things will be very very similar

like the Western blotting that means we

use antibody to tack to the protein

because we generally we cannot directly

add a add a fluoresent Dy to a protein

because a protein have a specific region

to bind so we develop certain antibody

which has the binding section to that

interest protein of our interest so that

that antibody can direct Bine now that

antibody can itself carry a that

antibody is called primary antibody now

that can carry a floresent tag right or

that may not carry a floresent tag we

need to have another secondary antibody

to be B bound with that which carries

the floresent tag in either way either a

single antibody which will detect the

protein may also have the floresent tag

may also carry the floresent tag in that

case we need only one antibody for the

reaction to detect the specific region

of modification or in other case we may

need two different antibodies first

antibody is simply bind to our desired

protein second antibody will bind to the

first antibod that will contain the tag

right uh if you are confused about

primary and secondary antibody I

recommend you to watch my video on

primary and secondary antibody you'll

find a different video completely

different separate video on primary and

secondary antibody you can watch that

video in the YouTube channel just search

it and you can find it and learn it but

the idea here the difference between the

B basic Western blotting and Eastern

blotting is that in this case of Eastern

blotting we

use uh the proteins and the antibodies

that we use they are targeted towards

the modification sites of proteins not

rest of the region of the protein

because in in Western blotting we target

specific sections of amino acid of the

protein but in this case we only target

modified amino acid of the protein

modified regions of the protein for

example a protein for example let's say

protein may have uh let's say

phosphorilation not phosphor let's say

may have a methylation out there now we

develop an

antibody that will tag or that will bind

to the methylated domain of that protein

only it will not bind any other place of

the protein remember this is very very

important so to design and find the

specific antibody which will only bind

with the modified section of the protein

is the key difference between eastern

blotting and western blotting okay rest

of the things are the same so that is

the thing that is the channel challenge

we need to find and we need to figure

out production of that antibody and

actually we can produce different types

of antibody if we know the sequence of

the protein we need to bind if we can

figure that out we can make a specific

antibody specific against that region of

the protein in the faab portion of that

antibody right because faab portion is

the portion to bind with uh the specific

proteins so you can prepare it any time

if you want right so this is the process

of Eastern blotting and that's how the

easn blotting is done very very simple

just like any other blotting but here

the proteins and the antibody that we

target will Target against the modified

region only okay so that's it guys if

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