Western vs. Southern vs. Northern Blots: Key Differences

Western vs. Southern vs. Northern Blots: Key Differences

Blotting techniques are one of the commonly used techniques in molecular biology and life sciences labs to detect and study different molecules, such as proteins and nucleic acids. The term “blotting” refers to the transfer of biomolecules to a membrane. 

Based on the molecules separated, such as DNA fragments, RNA, or proteins, the blotting method has three different types:

  • Western blotting
  • Northern blotting
  • Southern blotting

During the process, the molecules separated using the gel electrophoresis technique in the gel matrix are transferred to blotting membranes, such as nitrocellulose membranes and PVDF membranes. The transferred bands are visualized using different detection methods. 

The blotting techniques have applications in many research studies, including gene expression, hybridization, and identifying target sequences, such as specific DNA sequences or specific RNA sequences. 

Figure: Different types of blotting techniques for identifying different macromolecules. 

In this article, we will review more about different types of blotting techniques, their working mechanism, and applications in labs and industries.  

Defining Blotting Techniques

All three blotting techniques, western, southern, and northern blotting, separate different types of macromolecules and involve different reagents in the process.  

Western Blotting

Western blotting technique is also known as immunoblotting. The method was developed by Towbin and his colleagues in 1979 to study specific proteins. 

The protein blotting procedure involves four stages:

  1. Protein separation: Physical or chemical methods are used to break cell membranes and isolate desired proteins.
  2. Gel electrophoresis: SDS-PAGE is the most commonly used technique to separate proteins based on their molecule weight or charge from a sample mixture. The proteins are separated in the form of bands in the gel matrix. To visualize the proteins while being separated, dyes like bromophenol blue are used. 
  3. Blotting and transfer: The blotted proteins are transferred to either the nitrocellulose membrane or PVDF membrane through simple diffusion or electrophoretic elution.
  4. Visualization: The transferred bands are then visualized by using radiolabeled antibodies. Two types of antibodies used in the process include primary antibodies (specific to the target protein) and secondary antibodies (specific to the primary antibody).

The secondary antibody is conjugated either to an enzyme, fluorescent proteins, or biotin that forms a color change or fluorescence when a suitable substrate is added to the membrane. 

Figure: Western blotting steps.

Southern Blotting 

Southern Blotting techniques as introduced by Edwin Southern in 1975. The technique detects and analyzes a range of DNA fragments or DNA sequences in a whole DNA sample. The steps involved in the technique include:

  1. Isolation: DNA is isolated from cells by breaking the cell membrane using detergents and removing contaminants such as proteins using enzymes like proteinases. 
  2. Cleavage: Restriction endonuclease is used to break down the isolated DNA into small fragments and identify them using DNA probes. 
  3. Separation: The DNA fragments are separated using PAGE (Polyacrylamide gel electrophoresis) or agarose gel electrophoresis based on their molecular weight. 
  4. Denaturation: Alkaline solutions like NaOH are used to denature separated DNA. 
  5. Transfer: The DNA bands are transferred on a nylon or nitrocellulose membrane and made permanent by exposure to UV rays. 
  6. Hybridization: A labeled hybridization probe (a DNA fragment with a homologous sequence to the target DNA) is used to bind to the membrane to initiate a measurable reaction. 
  7. Visualization: For the visualization of separated bands, a dye such as EtBr (ethidium bromide) is added to the DNA sample so the fragment glows when observed under UV light. 
Figure: Southern blotting procedure.

Northern Blotting

The technique is used to detect and analyze specific RNA molecules from a mixed RNA sample. The technique was developed by Alwin in 1979 to study known mRNA sequences. The steps involved in the blotting procedure include:

  1. RNA separation: RNA is isolated from the source by cell lysis and removing DNA and protein contaminants using enzymes such as protease and DNAse. 
  2. Designing complementary DNA: A cDNA having a complementary sequence to the RNA of interest and labeled with either a luminescence or radiologically active probe is designed. 
  3. Gel electrophoresis: RNA fragments are separated in the gel matrix based on their length or size using the agarose gel electrophoresis technique. Here, the secondary structure of RNA is broken down into a primary linear structure by using formaldehyde. 
  4. RNA blotting: The separated RNA is transferred from the gel matrix to an aminobenzoxymethyl (ABM) filter paper by using the blotting technique. 
  5. Probe addition: A probe designed specifically for RNA of interest is added to the membrane that forms a DNA-RNA hybrid. This is followed by washing the unhybridized probe multiple times using many changes of buffer. 
  6. Detection: If the bands are hybridized using a radiolabeled probe, they generate a signal or light when exposed to X-ray film. This helps in visualizing and measuring the band intensity to determine the amount of molecules of interest.  
Figure: Northern blotting procedure.

Other than these, some other immunoblotting techniques (less commonly used) are also known, such as eastern blotting and southwestern blotting assays. 

Western vs. Southern vs. Northern Blotting

Here’s a table to help you clearly distinguish all three, western, northern, and southern blotting techniques side by side:

So, while experimenting, consider what type of biomolecule you want to analyze using the technique, whether it’s a protein or nucleic acid. This will help you to determine which method is suitable for your lab workflow. 

Boost Your Molecular Insights With Excedr’s Blotting Solutions

Blotting techniques are methods that involve transferring the separated biomolecules in a gel matrix to the membrane filter, such as nylon, nitrocellulose, or PVDF membrane. The technique is used in many life sciences labs, such as biotechnology and biochemistry for the analysis of a range of biomolecules, such as proteins and nucleic acids. 

Three commonly known and extensively utilized blotting techniques include western blotting (for protein analysis), northern blotting (for RNA studies), and southern blotting used for DNA sample analysis.

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