Special techniques

Affinity purification of antibodies from blots

Small amounts of specific antibodies can be recovered from excised bands of a blot. The antibodies can serve as reagents in immunohistochemistry or for clarifying suspected cross-reactions between protein mixtures. In order to deposit a large amount of antigen on the blot the sample is applied across the whole width of a slab gel.

Protein sequencing

In this technique proteins are transferred from SDS-gels to glass fiber filters or membranes resisting the organic solvents and reagents used for sequencing. Proteins, visualized by a general protein stain, are excised and directly inserted into an automatic sequencer. The technique is used for small amounts of protein which are difficult to purify by other methods.

Ligand blotting

Many solubilized receptor proteins will specifically bind their ligands and do so even after western blotting if harsh treatments (heating in SDS, reduction of disulfide bonds) are avoided at all steps. The ligand-receptor pairs analyzed by this procedure encompass a wide variety of molecules (steroids, peptide hormones, toxins, adhesion molecules). Even live cells (bacteria, eukaryotic cells) have been found to interact specifically with immobilized blotted adhesion proteins.

Fingerprinting of pathogens

This is useful for epidemiological studies of bacteria and viruses. Pathogens are cultured by usual microbiological techniques and isolates are subjected to SDS-gel electrophoresis and blotting. The strips are incubated with a standard antiserum and developed with suitable secondary reagents. The banding pattern characterizes the strain and distinguishes it from other strains.

Immunization

Proteins attainable only in small amounts are difficult to purify by liquid chromatography but may be separated by gel electrophoresis. The small amounts of pure proteins in a resolved band often suffice to immunize experimental animals. As an alternative to homogenizing the relevant slices of polyacrylamide (which leads to quite voluminous slurries) the blotted protein band may be implanted or injected into an animal.

Dot immunobinding

Other names for dot immunobinding are dot blotting and dot assay. The high binding capacity of blotting membranes can also be utilized for immunoassays where the electrophoretic separation of western blotting is not required. The antigen solution is spotted on to the membrane as a drop of 1 julI volume or smaller, resulting in a dot of a few millimeters diameter. Larger volumes may be deposited in a filtration manifold where proteins adsorb to the membrane. The technique is useful for adsorbing relatively impure antigens where the reactive components only constitute a small fraction of the total protein. The investigator is free to deposit different antigen and control preparations on the same piece of membrane. Further processing for antigen detection is analogous to western blotting.

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