Blotting Techniques

Blotting Techniques mainly have four main steps

• Electrophoretic separation of protein/nucleic acid fragments in the sample
• Transfer to and immobilization on a solid support
• Binding of probe to the target molecule on membrane
• Visualization of bound probe

Southern blotting – Allows DNA fragments to be identified

Northern blotting – Allows RNA fragments to be identified

western blotting – Allows proteins fragments to be identified

Southern blotting

1. Purification of DNA
2. Restriction enzyme digestion
3. Electrophoresis of the DNA
4. Denaturation and Neutralization of the DNA
5. Transfer of the DNA to a solid support – Capillary transfer, electrotransfer, and vacuum transfer
6. Prehybridization (blocking) – Wash the nylon/nitrocellulose membrane with a prehybridization buffer solution containing salmon sperm DNA, prevents non-specific attachments of a probe to the membrane
7. Hybridization with a labeled probe – Radioactively labelled, Florescent labelled Enzyme labelled
8. Visualization

Northern blotting

1. Purification of RNA
2. Electrophoresis of the RNA – agarose gel with formaldehyde as the denaturing agent ‒ limits secondary structures of RNA molecule
3. Transfer RNA to a solid support
4. Prehybridization (blocking)
5. Hybridization with labeled probe
6. Visualization