Loading controls are required to check that the lanes in your gel have been evenly loaded with sample. For most runs, it is convenient to reserve at least one separate lane on the gel to run the molecular weight markers. 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. Molecular weight markers are used to define the size of proteins run in a gel. A standard migration buffer (also called running buffer) for PAGE is 1 X Tris-glycine. The gels will be submerged in migration buffer which normally contains SDS, except in native gel electrophoresis. Load 20-40 μg total protein per mini-gel well. Never overfill wells, this could lead to poor data if samples spill into adjacent wells and poorly resolved bands. Take care not to pierce the base of the well with the tip as this will create a distorted band. Use special gel loading tips or a micro-syringe to load the complete sample in a narrow well. In addition, we usually add 0.1mg bromophenol blue as indicator into the sample loading buffer. 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. 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. Sample Loading of Western Blot Loading buffer: Learn more about western blot, please see Previous Section.
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