The technique uses polyacrylamide gel electrophoresis to separate denatured proteins by the length of the polypeptide, or (less commonly) native proteins by 3-D structure. The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are often detected with antibodies specific for an epitope in the target protein, or specific for a particular post-translational modification of an epitope.
There are a number of challenges here, leading to a range of false positive or false negative outcomes purely because of the laws of physics. For example, all proteins with the same molecular weight will bunch together during gel electrophoresis. Similarly, as proteins are transferred from the gel to the membrane some local (or even global) refolding might occur. The nature and extent of this is dictated by the physiochemical properties and primary sequence of the protein itself, by the buffer (if any) through which it moves, the heating of the system as an electrical field is applied across multiple layers of material with differing resistances and by all combinations of these variables. Any refolding event may re-create pre-existing conformational epitopes, destroy linear epitopes recognised by many antibodies, mask an epitope or even create novel, biologically irrelevant epitopes.
This leads to significant problems. For example, when labs try to compare western blotting results but use different blotting systems (dry, semi-dry, wet etc) for the transfer, it will be almost impossible to replicate each others findings. Similarly, cell biologists very often want to be able to detect a protein in vitro by western blotting and inside cells, using techniques such as immunofluorescence. Ideally the same antibody would be used for both experiments – and indeed one of the standard controls before using an antibody in immunofluorescence is to make sure that it recognises a single band of the correct molecular weight in a western. But if the transfer protocol has led to loss of cross-reacting epitopes and false negative outcomes, what is thought to be a reliable antibody on the basis of the western blot will actually yield misleading results in immunofluorescence. This means that on top of the frustrating reality that many antibodies which work in western blots fail to give a signal in immunofluorescence, even one that has been extensively validated by westerns must be taken with a pinch of salt. The result can only be validated after independent replication using at least one other antibody, by showing that the immunofluorescence signal goes away following successful RNAi-mediated knock-down of the target protein, or using a GFP-fusion to the protein.
Although there are a number of physical factors that can affect the western blot process, biology is even more messy. Antibodies are often used as the probes in the western blot process, and will benefit from the fact that the epitopes they recognise are being presented in much the same way as they would be in vivo – linear epitopes presented by T cells or by MHC complexes on target cells would be reflected by the denatured proteins recovered from gel electrophoresis, while the repeated epitopes typically presented by pathogen coats will also be found in bands on a membrane containing millions of copies of the target protein. This allows the same increases in observed binding affinities through avidity. All of this means antibodies should perform better than other probes in western blotting. However, the well-known non-specific reactivity of an antibody can lead to false positives by cross-reacting with a protein band at the same location as the true target, especially when probing for common post-translational modifications. False negatives may be caused by physical loss of a conformational epitope or by post-translational modifications that mask the epitope.
Building a successful assay is then down to the scientist, and those pesky laws of physics!
For more information about why Affimer technology are the first choice when looking for a custom affinity reagent contact us.