34
Semi-Dry Blotting of E-PAGE
™
Gels,
Continued
Semi-Dry Blotting
Protocol
1.
In a clean container or Incubation Tray, soak 4 pieces of 2.5 mm Blotting
Filter Paper (8.6 cm x 13.5 cm) in 2X NuPAGE
®
Transfer Buffer (see
page 32). Remove any air bubbles trapped between filter paper sheets using
the Blotting Roller while the paper is still submerged in buffer.
2.
In a clean container or Incubation Tray, soak the E-PAGE
™
Blotting Pad in
2X NuPAGE
®
Transfer Buffer (see page 32). Inspect both sides of the pad for
air bubbles. Press the pad to ensure the elimination of any visible air
bubbles.
Note:
The Blotting Pad has no specific orientation (either side may be facing
toward the gel).
3.
Place 2 pieces of pre-soaked 2.5-mm Blotting Filter Paper from Step 1 on the
anode plate of a semi-dry blotting apparatus. Ensure that all filter paper
sheets are aligned properly and remove any air bubbles with the Blotting
Roller.
4.
Place the pre-soaked blotting membrane (see page 32) on top of the filter
paper stack and remove any air bubbles with the Blotting Roller.
5.
Remove the gel from the transfer buffer. Gently rub a gloved finger over the
well side of the gel to remove small gel pieces from the gel surface. Re-
submerge the gel in transfer buffer to remove any gel pieces from the gel, as
they can cause air bubbles and field distortion during transfer.
6.
Place the flat side of the gel on top of the blotting membrane (well side up),
and remove any air bubbles with the Blotting Roller. Fill the wells of the gel
with 2X NuPAGE
®
Transfer Buffer (see page 32).
7.
Place the pre-soaked E-PAGE
™
Blotting Pad on the gel and gently roll out
air bubbles with the Blotting Roller.
8.
Place 2 pieces of pre-soaked 2.5-mm Blotting Filter Paper from Step 1 on top
of the Blotting Pad. Ensure that all filter paper sheets are aligned properly
and flush with the gel/membrane sandwich. Remove any air bubbles
trapped between filter paper sheets using the Blotting Roller.
9.
Place the cathode plate on the stack without disturbing the blot sandwich.
Follow the manufacturer’s instructions to further assemble the semi-dry
blotting apparatus.
10.
Transfer at 25 V for 1 h (~19 V/cm). You may need to optimize the transfer
conditions for your specific proteins or semi-dry blot apparatus. See
page 53 for results obtained with semi-dry blotting of E-PAGE
™
Gels.
Summary of Contents for E-PAGE Gels
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