um TE70X-IMA0
•
p18
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.
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