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.