Biomedical Engineering Reference
In-Depth Information
Removal by precipitation
Addition of potassium salts precipitates SDS nearly quantitatively,
but there is the risk of co-precipitation proteins.
References
Henderson LE, Oroszlan S, Konigsberg W (1979) Anal Biochem 93:153
Hjelmeland ML (1990) Meth Enzymol 182:277
2.5.3 Electrotransfer of Proteins onto Membranes
(Electroblotting; Western Blot): Semi-dry Blotting
The electrotransfer of proteins onto (non-specific) binding mem-
branoussheetsisnamedWesternblotincontrasttothetransfer
of DNA (Southern blot) and of RNA (Northern blot). The main
advantage of blotting procedures lies in the immobilization and
presentation of macromolecules on the surface of a solid planar
material. This presentation leads to an easy access of reactants in
the opposite to the diffusion-controlled motion of reaction partners
within gels or macroporous spheres.
Whereas gels are mainly electroblotted immediately after elec-
trophoresis, it is possible to blot Coomassie or Amido Black stained
gel too, but with lower efficiency and after soaking in ddH
2
Ofor
15 min and an equilibration step in transfer buffer C (see below)
for 30 min.
Synthetic or semi-synthetic porous sheets, so-called mem-
branes, able to bind proteins and other macromolecules non-
specifically and with high capacity (amount of bound macro-
molecules per area), are mostly used as materials for planar blotting
Blotting supports
supports. The most applied material for protein blotting are nitro-
cellulose (NC) and polyvinylidendifluoride (PVDF) and with some
less extended neutral or positively charged nylon.
Proteins interact with the membrane (support) by hydrophobic
and charge-transfer forces and hydrogen bridges. The extent of
these interactions depends on the accessibility of respective area of
a protein. The accessibility is influenced, among other things, by
the composition of the surrounding buffer, e.g., pH, ionic strength
and/or chaotropic additives.
Two technical variants of electrotransfer from PAGE gels to
membranes have been developed: the tank blotting developed by
Towbin and the semi-dry blotting proposed by Kyhse-Anderson.
With respect to transfer performance both systems are similar
but the semi-dry system is easier to handle and less material-
consuming; therefore, we followed the Kyhse-Anderson protocol
for blotting from SDS PAGE gels in our lab.
The semi-dry blotting apparatus consists of two plates (anode
and cathode) made from graphite, glassy carbon, or stainless steel
and a sandwich consisting of buffer-soaked filter paper, transfer
