Difference between Southern and northern blotting and western blotting

Summary

This video tutorial from 'Somos Biology' succinctly explains the three major blotting techniques: Southern, Northern, and Western blotting. Southern blotting detects DNA using agarose gel and a capillary transfer method with a DNA probe. Northern blotting, similar but for RNA, employs formaldehyde in the gel to linearize RNA and uses a DNA-RNA hybridization probe. Western blotting is distinct, detecting proteins with a polyacrylamide gel and electroblotting, utilizing antibodies as probes. The video clarifies differences in sensitivity, electrophoresis, blotting methods, probes, and detection systems, inviting viewers to explore each technique in greater detail through linked videos.

Takeaways

• 🔬 **Blotting Techniques Overview**: The script introduces three major types of blotting techniques used to detect macromolecules within cells: DNA, RNA, and protein molecules.
• 🧬 **Southern Blotting**: This technique is used for detecting DNA molecules and is named after its discoverer, E.M. Southern.
• 📚 **Northern Blotting**: Named after Southern's technique, it is used to detect RNA molecules and involves resolving secondary structures with formaldehyde in agarose gel.
• 🥼 **Western Blotting**: Named in the direction of 'Southern' and 'Northern', this technique detects protein molecules and is the most sensitive among the three.
• 🚫 **No Directionality**: Despite the directional names, the blotting techniques are not related to any directional process.
• 🔬 **Electrophoresis Types**: Southern and Northern blotting use agarose gel, while Western blotting uses polyacrylamide gel (SDS-PAGE) for separation.
• 🌊 **Blotting Methods**: Southern and Northern blotting use capillary action for transferring molecules to a membrane, whereas Western blotting uses electroblotting.
• 🧪 **Probes Used**: Southern blotting uses single-stranded DNA probes, Northern blotting uses cDNA probes for DNA-RNA hybridization, and Western blotting uses antibodies.
• 🔍 **Detection Systems**: Southern and Northern blotting can use radioactivity, chemiluminescence, or colorimetric analysis, while Western blotting typically uses chemiluminescence or colorimetric analysis.
• 🔗 **Further Learning**: The script encourages viewers to watch more detailed videos on each blotting technique for a deeper understanding of the processes and techniques.
• 👍 **Engagement Call**: The video ends with a call to action for viewers to like, subscribe, and share the video, emphasizing the importance of sharing knowledge.

Q & A

What are blotting techniques used for?

Blotting techniques are used for detecting macromolecules such as DNA, RNA, or protein molecules present inside the cell at a specific time.

What is the purpose of Southern Blotting?

Southern Blotting is used to detect DNA molecules.

How is Northern Blotting different from Southern Blotting?

Northern Blotting is used to detect RNA molecules, while Southern Blotting is used for DNA molecules.

What is the primary purpose of Western Blotting?

Western Blotting is used to detect the presence of protein molecules.

Why are the names Southern, Northern, and Western Blotting used, and do they have any relation to directionality?

The names are derived from the discoverers of the respective techniques: E.M. Southern for Southern Blotting, and they do not have any relation to directionality.

What is the most sensitive blotting technique among the three?

Western Blotting is the most sensitive, as it can detect up to the nanogram level of proteins.

What type of gel is used for electrophoresis in Southern and Northern Blotting?

Agarose gel is used for electrophoresis in both Southern and Northern Blotting.

What is the difference in the gel used for Western Blotting compared to Southern and Northern Blotting?

Western Blotting uses polyacrylamide gel (SDS-PAGE) instead of agarose gel.

How is the blotting process carried out in Southern and Northern Blotting?

In Southern and Northern Blotting, the capillary method is used for transferring the DNA or RNA from the gel to the membrane.

What technique is used for protein transfer in Western Blotting?

In Western Blotting, electroblotting is used to transfer proteins from the gel to the membrane using an electric current.

What types of probes are used in Southern, Northern, and Western Blotting?

Southern Blotting uses single-stranded DNA probes, Northern Blotting uses cDNA probes for DNA-RNA hybridization, and Western Blotting uses antibodies (primary and secondary).

What are the detection systems used in these blotting techniques?

Southern and Northern Blotting can use radioactivity, chemiluminescence, or colorimetric analysis, while Western Blotting typically uses chemiluminescence or colorimetric analysis due to the difficulty of radiolabeling proteins.

Outlines

00:00

🧬 DNA, RNA, and Protein Detection Techniques

This paragraph introduces the three major types of blotting techniques used for detecting macromolecules within cells: DNA, RNA, and proteins. Southern blotting is used for DNA detection, Northern blotting for RNA, and Western blotting for protein detection. The names are derived from their discoverers and do not imply directionality. The paragraph outlines the similarities and differences between the techniques, highlighting their sensitivity levels and the types of molecules they target.

05:01

🔍 Detailed Comparison of Blotting Techniques

This paragraph delves deeper into the specifics of each blotting technique. Southern and Northern blotting are similar in that they both use agarose gel for electrophoresis, with Northern blotting incorporating formaldehyde to linearize RNA. Western blotting, however, uses polyacrylamide gel. The blotting methods differ as well, with capillary action used for Southern and Northern blotting and electroblotting for Western blotting. The probes and detection systems vary, with DNA probes used for Southern blotting, DNA-RNA hybridization for Northern blotting, and antibodies for Western blotting. Detection methods include radioactivity, chemiluminescence, and colorimetric analysis, with the latter two preferred for Western blotting due to the difficulty of radiolabeling proteins.

Keywords

💡Blotting Techniques

Blotting techniques are laboratory methods used to transfer and detect specific molecules, such as DNA, RNA, or proteins, from a gel to a membrane. In the video, the main theme revolves around three major types of blotting techniques, each designed to detect a different macromolecule, and the script explains how these techniques are essential for molecular biology research.

💡Southern Blotting

Southern Blotting is a specific blotting technique named after its inventor, E.M. Southern. It is used to detect the presence of a specific DNA sequence. The script mentions that it involves using agarose gel electrophoresis to separate DNA molecules and then transferring them to a nitrocellulose membrane for detection with a DNA probe.

💡Northern Blotting

Northern Blotting is another blotting technique, used to detect RNA molecules. The script explains that it is similar to Southern Blotting but uses a formaldehyde-agarose gel to resolve the secondary structure of RNA, allowing separation based on length. The detection involves a cDNA probe, which hybridizes with the target RNA to form a DNA-RNA hybrid.

💡Western Blotting

Western Blotting is the technique used for protein detection and is distinguished by its use of antibodies as probes. The script describes it as the most sensitive of the three techniques, capable of detecting proteins in the nanogram range. It uses a polyacrylamide gel (SDS-PAGE) for protein separation and an electroblotting technique to transfer proteins onto a membrane.

💡Molecular Detection

Molecular detection refers to the process of identifying and quantifying molecules within a sample. The video script emphasizes the role of blotting techniques in detecting specific macromolecules—DNA, RNA, or proteins—at a particular time, which is crucial for understanding cellular processes.

💡Electrophoresis

Electrophoresis is a technique used to separate molecules based on their size and charge. In the context of the video, different types of electrophoresis—agarose gel for DNA and RNA, and polyacrylamide gel for proteins—are mentioned as the second stage in blotting techniques, crucial for the separation of molecules before transfer to a membrane.

💡Blotting Process

The blotting process is the method by which separated molecules are transferred from the gel to a membrane. The script details different blotting methods, such as capillary blotting for DNA and RNA, and electroblotting for proteins, which are essential for the subsequent detection of the molecules.

💡Probe

A probe in molecular biology is a molecule used to detect and locate a target sequence in a sample. The video script explains that different types of probes are used in blotting techniques: single-stranded DNA for Southern Blotting, cDNA for Northern Blotting, and antibodies for Western Blotting.

💡Hybridization

Hybridization in the context of blotting techniques refers to the process where a probe binds to its complementary sequence in the target molecule. The script illustrates this with DNA-DNA hybridization in Southern Blotting and DNA-RNA hybridization in Northern Blotting.

💡Detection System

The detection system is the method used to visualize the presence of the target molecule after hybridization. The script explains that radioactivity, chemiluminescence, or colorimetric analysis can be used for Southern and Northern Blotting, while Western Blotting typically employs chemiluminescence or colorimetric analysis due to the difficulty of radiolabeling proteins.

💡Agarose Gel

Agarose gel is a type of gel matrix used in electrophoresis to separate molecules based on size. The script mentions its use in both Southern and Northern Blotting for the separation of DNA and RNA molecules, respectively.

💡Polyacrylamide Gel (SDS-PAGE)

Polyacrylamide gel, specifically SDS-PAGE, is used in Western Blotting to separate proteins based on their molecular weight. The script describes it as a different type of gel from agarose, which allows for the resolution of proteins in a way that is suitable for their detection.

Highlights

Introduction to the video tutorial on blotting techniques from Somos Biology.

Summary of the three major types of blotting techniques: Southern, Northern, and Western blotting.

Explanation of the purpose of blotting techniques: detecting macromolecules in the cell.

Southern blotting is used for DNA molecule detection.

Northern blotting is utilized for RNA molecule detection.

Western blotting is for protein molecule detection.

Clarification that the names of blotting techniques do not imply directionality but are named after their discoverers.

Description of the four stages involved in blotting techniques: extraction, electrophoresis, blotting, and probing.

Southern and Northern blotting are similar as they deal with DNA and RNA, while Western blotting is distinct.

Western blotting is the most sensitive technique, capable of detecting nanogram amounts of DNA.

Different types of electrophoresis used in blotting: agarose gel for Southern and Northern, and polyacrylamide gel for Western.

Use of formaldehyde in Northern blotting to resolve RNA secondary structures for linear separation.

Blotting methods vary: capillary method for Southern and Northern, and electroblotting for Western.

Different probes used in blotting: DNA probes for Southern and Northern, and antibodies for Western.

Hybridization processes differ: DNA-DNA for Southern, DNA-RNA for Northern, and antibody binding for Western.

Detection systems vary: radioactivity and chemiluminescence for Southern and Northern, chemiluminescence or colorimetric analysis for Western.

Recommendation to watch detailed videos on each blotting technique for further understanding.

Invitation to like, subscribe, and share the video for more educational content.

Transcripts

welcome again friends welcome to another

video tutorial from somos biology we've

been talking about different types of

blotting techniques in this video I will

be summarizing all the major three types

of blotting techniques and the

difference between southern blotting

northern blotting and western blotting

so let us go with that the blotting

technique means is the way of detecting

the macro molecules that are present

inside the cell at a specific time now

it could be DNA it could be RNA or

protein molecules now if we are going to

detect DNA molecule we use southern

blotting if you are going to detect RNA

we use northern blotting if you want to

detect protein go for western blotting

the name of these things are with

directions but there is no link with

directionality because the name of

southern blotting was was according to

the name of the discoverer of this

technique eeehm southern and then after

four years the other technique of RNA

detection is determined that is known as

northern and then as is these things are

named like southern and northern the

third and the last techniques that is

developed which is which is the

direction of protein came out to known

as the Western blot so now if you look

at here we'll be seeing some

characteristics for all these three and

the difference is between all these

three based on these characteristics

starts with the molecules detected in

the process of southern blotting we

detect DNA in the presence of DNA

molecule in case of northern blotting we

detect the presence of RNA molecule in

case of Western blotting we detect the

presence of protein molecule okay

now among these three blotting

techniques southern blotting in northern

blotting is very very similar because

they deal with DNA and RNA while Western

blotting is different from southern and

northern blotting western blotting is

the most sensitive among these three

because it can detect up to the nanogram

of the DNA okay

the second characteristic

the electrophoresis type that we use to

separate now in any blotting techniques

there are three different stages the

first stage is the extraction of the

molecule the second stage is the

electrophoresis or the separation of the

molecule and the third stage is a

blotting process and the fourth stage is

the probing of that molecule so there

are actually four different stages in

case of this electrophoresis southern

blotting where we do the separation of

DNA we use agarose electrophoresis or

agarose gel is used for the

electrophoresis purpose in case of

northern blotting we use agarose gel but

the Tejanos gel is carrying formaldehyde

because in case of northern blotting the

RNAs are normally extracted in the in

the secondary structure format so we

need to resolve the secondary structure

to make the RNA linear so that the

separation of RNA can be done based on

their length that is we use formaldehyde

in the agarose gel in case of northern

blotting in case of western blotting we

is completely different type of gel and

in this case we use is d s page which is

a poly acrylamide gel in case of

southern and northern we use agarose gel

but in case of western blotting use poly

acrylamide gel okay the third

characteristic is the blotting method

that we use the blotting method that we

use in southern blotting is is using a

capillary if using so it using capillary

force or capillary method that will help

to take the DNA component from the gel

binding towards the nitrocellulose

membrane in case of northern blotting we

also use this capillary capillary force

for transferring the content of RNA that

is present in the gel to the membrane

what we do here we simply put

Jayla on the bottom then we put the put

the capillary that the membrane and we

place them in the buffer so that buffer

can run and pass through the gel and

then finally to the membrane that is the

capillary force that we use in case of

Western blotting however we use glitter

electro illusion later illusion

technique in that case what we do we put

the gel and we put the membrane but we

put the electrodes which will help to

migrate the proteins we use current as

like electrophoresis

to transfer proteins from the gel to the

membrane that is the difference now the

look at the fourth characteristics that

is a probe that we use the probe that we

use in case of southern blotting our DNA

probe single-stranded DNA are used as a

probe which will be attached with any

kind of radio active or radio leveling

is done and also it could be attached

within some chemical molecules that is

attached to like any chemiluminescence

molecule in case of northern blotting we

also use DNA probe so the hybridization

process of the probe with the target in

case of DNA is dla in case of southern

blotting is DNA DNA hybrid but in case

of northern blotting the hybridization

is done by dna-rna hybrid because RNA is

the target and the probe that we use is

the cDNA

so the dna-rna hybrid is formed well in

case of western blotting is completely

different we use antibody as a probe and

actually use two different antibody

primary antibody and secondary antibody

in this whole process of probing and the

last one that is the detection system

the detection system that we use in case

of southern blotting and northern

blotting as are seen we use either

radioactivity

we can use chemiluminescence we can also

use color in a colorimetric analysis in

case of northern blotting we do the same

as I told you it is a Western blotting

radioactivity is not used and not

preferred because there is no way of

marking the proteins or radio labeling

the proteins in a convenient way so we

use either chemiluminescence or quality

metric analysis chemiluminescence means

after the reaction we get the light the

emission of light that can prove the

presence of the target protein or

colorimetric analysis can also give us a

generation of a color that can also

prove the presence of the protein so

that in a sense are the difference

between southern blotting northern

blotting and western blotting I hope

this video helps you to understand about

the process now if you want to talk

about and understand each of this

process in much more details with all

the steps and techniques I will

recommend you to watch my 3 videos on

both southern blotting northern blotting

and western blotting I made new videos

among all this type of blotting

techniques which you can watch anytime

and the links are provided in the

description so hope you like this video

if you like this video please hit the

like button subscribe to my channel to

get more videos like that and share this

video with your friends remember sharing

is caring thank you