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Semi-dry transfer has traditionally been a fast but inefficient method for transferring all the protein from a gel. By using a discontinuous buffer system, transfer efficiency can be greatly increased. In protein blotting, the buffer has an important effect on the elution of the proteins from a gel and the retention of the protein to the membrane.
A unique feature of semi-dry blotting is the ability to use two different buffers during transfer, known as a discontinuous buffer system. This is important because the effect of methanol and SDS are opposing to both the gel and membrane respectively. Optimization of the buffer with respect to both the gel and membrane can be accomplished using a discontinuous buffer system.
The Effects of Methanol
Methanol increases a proteins affinity for a membrane by removing the
SDS from the protein. This increases the available hydrophobic sites on
the protein for binding to the membrane support. It is for this reason
methanol is often used in transfer buffers. While advantageous for binding
of the protein to the membrane, methanol causes the pores in the gel to
constrict. This makes it mechanically more difficult for the protein to
exit the gel.
The Effects of SDS
SDS is used in western blotting transfer buffer to aid in the elution
of proteins from the gel matrix. While it inhibits the binding of the
protein to the membrane, it is often necessary to facilitate complete
transfer of proteins from the gel.
Using A Discontinuous Buffer System
The opposing effects of methanol and SDS in blotting can be exploited
in semi-dry
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