Biomedical Engineering Reference
In-Depth Information
membrane and electophoresis gel. The sandwich is made from
several layers of filter paper (three to four sheets of, for example,
Whatman MM3), the membrane, and the gel (Fig. 2.3). Filter paper
and membrane should have the same size as the gel.
Important!
Never touch gels or membranes with fingers. Wear
gloves and/or use tweezers!
A
first anode buffer
: 300 mM Tris in ddH
2
O (do not correct pH)
Solutions/
Reagents
21
B
second anode buffer
:25mM Tris in ddH
2
O (do not correct
pH)
ε
C
cathode buffer
:25mMTr i s , 4 0 mM
-aminocaproic acid (EAC,
6-aminohexanoic acid), 0.04% SDS
If smaller proteins shall be blotted, add up to 20% methanol to the
buffers A, B, and C.
The formation of the transfer unit starts with the anodic fil-
ter paper stack. Two to three sheets of filter paper are soaked in
buffer A and placed onto the anode. Air bubbles are removed using
Fig. 2.3.
Principle of semi-dry blotting
21
Kondo et al. simplified the buffers. They used a buffer of 25 mM
Tris, 192 mM glycine for the anodic side, and 25 mM Tris, 192 mM
glycine, 0.1% SDS as cathode buffer. We did not find significant differ-
ences between both buffer systems. (Kondo M, Harada H, Sunada S,
Yamaguchi T (1991) Electrophoresis 12:685)
