Biomedical Engineering Reference
In-Depth Information
a glass rod. Then two to three sheets follow soaked with Soln. B.
ThemembraneiswettedwithSoln.Bandplacedontopofthe
filter paper. When using the very hydrophobic PVDF membranes,
submerge the membrane in methanol (take care that the whole
membrane is wetted, i.e., the color changes from white to opales-
cent) for some seconds and transfer it immediately to Soln. B. Do
not leave the wet PVDF membrane on air, because it dries very
quickly.
The SDS-PAGE gel is agitated for few minutes in Soln. B and
then put onto the membrane. Some layers of filter paper soaked
in Soln. C finish the sandwich. The blotting apparatus is covered
with the cathode plate and the electrodes are plugged into the
power supply. Electrotransfer is performed with a constant current
of 0.8 mA
/
cm
2
of gel area for 0.5 - 2 h at RT. The voltage does not
exceed 15 V.
Multiple electrotransfer of up to three gels is possible. On top of
the filter papers soaked with Soln. B a wetted dialysis membrane is
placed and then the sandwich is repeated. Transfer conditions are
similar to the single sandwich except the total gel area taken into
account for calculation of applied current.
If the conditions of the receiving material (e.g., activated paper
instead of nitrocellulose) forbid the use of Tris-containing buffers,
Buffer
modifications
the following substitutes may be used:
A
:0.3M triethanolamine (M
r
149.2), pH 9.0; B
:25mM tri-
ethanolamine, pH 9.0; C
:25mM triethanolamine, 40 mM butyric
acid, pH 9.0 to 9.2.
Important!
Use ultrapure triethanolamine, free of primary amines!
Proteins blotted on PVDF can be subjected directly to sequence
analysis. Specialized protocols for this purpose are referred to, for
example, by Jungblut (1997).
References
Beisiegel U (1986) Electrophoresis 7:1
Jungblut P (1997) In Kamp RM, Choli-Papadopoulou T, Wittmann-Liebold
B (eds.) Protein structure analysis. Springer, Berlin, p. 215
Kyhse-Andersen J (1984) J Biochem Biophys Meth 10:203
Towbin H, Gordon J (1984) J Immunol Meth 72:313
2.5.4 Immunochemical Detection of Antigens After
Electrotransfer (Immunoblotting)
Macromolecules blotted onto membranes are detected very specifi-
cally and sensitively by antibodies. The prerequisite is the existence
of epitope(s) on the surface of blotted antigens after denaturation
during SDS-PAGE. Very often, even fragile epitopes (which are
