Bio-Rad Trans-Blot® Cell - Manual (Page 14)

8. Transfer buffer recommendations
Use only high quality, reagent grade methanol. Contaminated methanol can result in
increased transfer buffer conductivity, as well as poor transfer of macromolecules. Do
not reuse transfer buffers or dilute transfer buffers below recommended levels. Reuse of
transfer buffers is not advised, since these buffers have most likely lost their ability to
maintain a stable solution pH during transfer. Dilution of transfer buffers below their rec-
ommended levels is also not advised, since this will decrease their buffering capacity.
9. Voltage limits
Do not increase voltage settings beyond those indicated in Tables 3.1–3.4 for overnight
operation. Buffer conductivity must be close to the current listed and a current limit should
be set on the power supply. If overnight transfers at low voltages are ineffective for your
application, and higher voltages are necessary, transfer times must also be decreased.
Failure to do so may result in a potential safety hazard.
3.3 Buffer Formulation
All formulas provided below are for a total volume of 1 liter of buffer. Four liters of
buffer are required for the Trans-Blot cell.
DO NOT ADD ACID OR BASE TO ADJUST pH OF THE FOLLOWING
BUFERS. Methanol should be analytical reagent grade, as metallic contaminants in low
grade methanol will plate on the electrodes.
Note: Some pH electrodes will not perform a proper measurement for the pH of Tris
buffers. If the pH of the buffer is off, check to make sure the electrode is designed to
work with Tris buffers. If the pH electrode functions properly for Tris buffers and the
pH is below 8.0, remake the buffer.
1. 25 mM Tris, 192 mM glycine, 20% v/v methanol, pH 8.3
Mix 3.03 g Tris, 14.4 g glycine, and 200 ml of methanol; add distilled deionized water
(dd H
2
O) to 1 liter.
2. 25 mM Tris, 192 mM glycine, pH 8.3
Mix 3.03 g Tris and 14.4 g glycine; add dd H
2
O to 1 liter.
3. 48 mM Tris, 39 mM glycine, 20% v/v methanol, pH 9.2
Mix 5.82 g Tris and 2.93 g glycine in ddH
2
O, add 200 ml methanol
Bring to 1 liter with ddH
2
O
4. 48 mM Tris, 39 mM glycine, pH 9.2
Mix 5.82 g Tris and 2.93 g glycine
Add ddH
2
O to 1 liter
5. 10 mM NaHCO
3
, 3 mM NaCO
3
, 20% methanol, pH 9.9
Mix 0.84 g NaHCO
3
and 318 g Na
2
CO
3
in ddH
2
O, add 200 ml methanol
Bring to 1 liter with ddH
2
O
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