Cardinal Blots

After the Southern Blot was named after Edwin Southern’s 1975 published protocol, scientists went a bit wild with the naming convention. As such we have 4 fairly distinct protocols with significantly different applications and outcomes, all sharing similar names. I must say I can never keep them straight, so I’m writing this page to remind myself which once is which when someone asks me at a dinner party.

Northern Blot (or) RNA blot

The Northern Blot is an RNA analysis technique that allows for the identification of specific RNA sequences. It is generally performed on the total mRNA content of a biological sample in order to gain insight into the transcriptome. The transcriptome describes the level of genetic expression between the genome and the proteome. Since not everything that is transcribed is translated, gaining insight into the transcriptome allows a researcher to gain a deeper understanding of the regulation of protein expression. Abnormalities in the production of mRNA is a useful marker of disease or mutation events and can allow for the early detection of cancer.

The Northern blot does not allow for significantly better quantification than an agarose gel on its own, instead it provides qualitative insight into the RNA sequence. The agarose gel for a Northern blot differs from a traditional DNA Agarose Gel. With a thickness of just 1.5 mm, it is an extremely thin and delicate gel, reminiscent of the SDS-PAGE gel. However rather than a denaturing agent and detergent, the Northern blot gel traditionally contains formaldehyde. This helps limit the effect RNA secondary structures have on the passage of the RNA through the gel.

Alternatively an SDS-PAGE gel can be used in place of the agarose gel, with a traditional denaturing agent such as Urea or Beta-Mercaptoethanol.

Protocol: Northern Blot

Reverse Northern Blot:

Eastern Blot (or) Post-Translation Protein Blot

The Eastern blot is used to analyse changes to proteins that occur after translation. An Eastern blot might be used after a Western blot to learn more about the post-translational changes to proteins that have been identified. A lot of authors are fighting to have their protocol known as the official ‘Eastern Blot’ protocol. The most common technique involves the transfer of an SDS-PAGE gel onto a polyvinylidene fluoride or nitrocellulose membrane. Synthetically designed probes are used to identify phosphorylation, carbohydrates, lipids or other folding changes that may influence how the protein functions. Alternate Eastern blotting protocols involves the use of different transfer membranes such as lectin.

Another definition of Eastern blotting defines it by the use of an aptamer probe rather than antibody probe. An aptamer is a synthetic molecule that is structurally different from the traditional Y-shaped antibody molecule, yet is designed to bind a target in a similar fashion.

Protocol: Eastern Blot

Southern Blot (or) DNA Blot

Southern blotting is a DNA detection technique that has been completely replaced by variations of the Polymerase Chain Reaction in most areas of molecular biology. It continues to have some use in the detection of large (>1 kb) genes in genomic DNA purifications. Restriction enzymes are used to slice apart the DNA, which is run out on an agarose gel before transfer to a nitrocellulose membrane. Probes are added that bind to specific sequences. Southern blotting may be a useful technique to consider if you need to identify a target that is larger than the upper limit of your polymerase (>10 kb) but most limitations in PCR can be designed around.

Protocol: Southern Blot

Western Blot (or) Protein Blot

Western blotting is an excellent and widely used Protein detection technique that follows naturally from an SDS-PAGE and can be used as an alternative to ELISA for identifying specific proteins. Proteins are denatured and separated by size-based electrophoresis on an SDS-PAGE. The proteins in the gel are then transferred to a nitrocellulose membrane, which is washed with a primary antibody specific to the protein(s) of interest. The gel is then washed with a secondary antibody with an attached fluorescent marker that binds to the primary antibody. The gel is then imaged, with the fluorescent marker allowing for the qualitative identification of the proteins already separated based on size by the SDS-PAGE.

Protocol: Western Blot