Transfer of proteins and nucleic acids on to the surface of immobilising membranes
- Protein blotting
- Nucleic acid blotting
- Sample concentration/immobilisation
Blotting is a standard method for the transfer of molecules on to the surface of a membrane. Additionally blotting is an intermediate step in protein sequencing and an elution method for subsequent analysis.
Electro-blotting is an important method to transfer mainly proteins from polyacrylamide gels on to nitrocellulose or other carrier membranes (Western blotting). Tank blotting is done in vertical buffer tanks with coiled platinum wire electrodes on two sides. For this technique, the gel and blotting membrane are clamped in a frame between filter paper and fiber pads. Typical transfer time is over night. Semi-dry blotting is done between two horizontal plate electrodes and offers fast as well as homogeneous transfer. In contrast to tank blotting only little transfer buffer is required and transfer times are very short (several minutes). Additionally, with semi-dry blotting discontinuous buffer systems can be used to gently blot smaller proteins or to transfer protein mixtures of very different sizes more evenly.
Transfer of nucleic acids (Southern / Northern blotting) is traditionally done by capillary blotting, a time-consuming procedure that usually takes up to 12 hours (over night) to complete. Therefore vacuum blotting is mostly used instead of capillary blotting. With vacuum blotting transfer times can be reduced to 15 - 60 minutes.
Dot blotting techniques are used for concentration and immobilisation of samples (proteins and nucleic acids) on to a membrane with the use of a vacuum source.