define western blot

1. Electrophoresis & Protein Transfer Laemmli (Tris-glycine) buffering systems are the most commonly used and are comprised of a stacking gel of pH 6.8 and a resolving gel of between pH 8 and 9. One potential drawback of this popular system is that disulfide bonds tend to form between cysteine residues at this relatively high pH, although this problem can be alleviated by the addition of a reducing agent to the sample. In addition, we usually add 0.1mg bromophenol blue as indicator into the sample loading buffer. Use special gel loading tips or a micro-syringe to load the complete sample in a narrow well. Take care not to pierce the base of the well with the tip as this will create a distorted band. Never overfill wells, this could lead to poor data if samples spill into adjacent wells and poorly resolved bands. Load 20-40 μg total protein per mini-gel well. The gels will be submerged in migration buffer which normally contains SDS, except in native gel electrophoresis. A standard migration buffer (also called running buffer) for PAGE is 1 X Tris-glycine. Molecular weight markers are used to define the size of proteins run in a gel. Markers are composed of different proteins of known size and the distances migrated over the time course of the run provide a logarithmic scale by which to estimate the size of unknown proteins. For most runs, it is convenient to reserve at least one separate lane on the gel to run the molecular weight markers. http://www.creative-diagnostics.com/Electrophoresis-Protein-Transfer.htm