Hoefer TE70X - Manual (Page 25)

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p17
5. Electrotransfer notes
• Run the transfer as soon as possible after
electrophoresis to minimize protein diffusion
within the gel.
• Stacked gels must all be the same size.
• Limit transfers to two hours or less.
• The recommended methanol concentration for
different membrane types are:
membrane type methanol %
Charged nylon 0
Nitrocellulose 10 – 20
PVDF 10 – 20
• Use a buffer with low ionic strength such as
one of the two listed below to prevent over-
heating. Use the CAPS buffer when Tris cannot
be used (e.g., peptide sequencing). CAPS can
improve transfer because of its effect on the
charge of the protein (see Matsudaira, 1987).
Towbin buffer
(25 mM Tris, 192 mM glycine, 20% v/v methanol,
pH 8.3, 1 liter)
Tris (FW 121.1)
25 mM 3.0 g
Glycine (FW 75.07) 192 mM 14.4 g
SDS* (FW 288.4) 0.1% (3.5 mM) 1.0 g
Dissolve in 600 ml distilled water.
Add methanol as required
† .
Bring to 1 liter with distilled water. Do not adjust the
pH, which should be between 8.2 – 8.4.
Optional: Chill before use.
*Optional: Adding SDS can improve transfer efficiency.
†
Depending on the membrane type selected (see table above), adding
methanol can improve transfer results.
Note: Buffers containing methanol
may deteriorate if stored for long
periods — add methanol just prior
to transfer.