4.3 Assembly of the Unit for Acidic Transfers
If an acidic transfer buffer is used, the transfer direction will be from the anode to the
cathode.
1. Remove the safety cover and the stainless steel cathode assembly.
2. Place a pre-soaked sheet of extra thick filter paper onto the platinum anode. Roll out all
air bubbles. If thin filter paper is used, repeat with two more sheets of buffer-soaked
filter paper. If thick filter paper is used, repeat with one more sheet of buffer soaked
filter paper.
3. Carefully place equilibrated gel on top of the filter paper, aligning the gel on the center of
the membrane. Roll out all air bubbles.
4. Place the pre-wetted blotting media on top of the gel. Roll out all air bubbles.
5. Place another sheet of pre-soaked extra thick filter paper on top of the blotting membrane,
carefully removing all air bubbles. If thin filter paper is used, place three sheets on top of the
membrane, or if thick filter paper is used, place two sheets on top of the membrane.
6. If more than one gel is to be transferred, place a sheet of pre-soaked dialysis membrane
on top of the filter paper stack. Repeat the procedure from step 2.
7. Carefully place the cathode assembly onto the stack. Press to engage the latches with the
guide posts, without disturbing the filter paper stack.
8. Place the safety cover on the unit. Plug the unit into the power supply, red wire to red
outlet and black wire to black outlet.
Caution: Do not reverse polarity. This will damage the stainless steel cathode.
9. Turn on the power supply. Transfer mini gels for 15–30 minutes at 10–15 V. Large gels
can be transferred for 30 minutes to 1 hour at 15–25 V. Do not exceed 25 V with this
instrument. A current limit (3 mA/cm
2
for large gels; 5.5 mA/cm
2
for mini gels) is
recommended to prevent excessive heating during the run.
Section 5
Buffer Formulation
The following buffers are recommended for use with the Trans-Blot SD cell. For protein
transfers, the single buffer system of Bjerrum and Schafer-Nielsen
16
provides more efficient
elution than the original isotachophoretic system of Khyse-Andersen, which requires the use
of three different buffers.
15
A carbonate buffer has also been shown to produce high
efficiency transfers with improved antibody recognition.
1. Bjerrum and Schafer-Nielsen transfer buffer for SDS-proteins using nitrocellulose (with
methanol) or Zeta-Probe membrane (without methanol):
16
48 mM Tris, 39 mM glycine, (20% methanol) pH 9.2
Dissolve 5.82 g Tris and 2.93 g glycine [and 0.375 g SDS or 3.75 ml of 10% SDS] in dd
H
2
O (add 200 ml of methanol); adjust volume to 1 liter with dd H
2
O.
DO NOT ADD ACID OR BASE TO ADJUST pH. The buffer will range from pH 9.0
to 9.4, depending on the quality of the Tris, glycine, dd H
2
O, and methanol. Methanol
should be analytical reagent grade, because metallic contaminants in low grade methanol
will plate on the electrodes.
10
(32 pages)
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