Proteins are most often separated by acrylamide gel electrophoresis. As these gels are too restrictive for capillary blotting, Western Blots are done by electro transfer.
Depending on the type of blotting (tank or semi-dry blotting) and the specific characteristics of the proteins, different buffers may be used for western blotting. For small proteins or for tank blotting after SDS PAGE Towbin Buffer is recommended, for blotting proteins after SDS PAGE or IEF by semi-dry blotting, a discontinuous buffer system is recommended.
A fast and easy to use alternative for optimal semi-dry blotting is SERVA’s Xpress Blotting Kit. The buffer system enables in combination with the newly developed alternative for blotting paper, the Blotting Fleece, an efficient simultaneous transfer of high and low molecular weight proteins in only 15 minutes.
Although nitrocellulose membranes are still used for routine applications, the optimum result is achieved with PVDF membranes.
Detection of membrane bound antigens or nucleic acid sequences labelled with Horseradish Peroxidase (HRP) or Alkaline Phosphatase (AP) can be done with colorimetric substrates like TMB or BCIP/NBT or chemiluminescent substrates.