ThermoFisher Scientific E-PAGE Gels, Техническое руководство

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36
Semi-Wet Blotting of E-PAGE
™
Gels,
Continued
Preparing 1X
Transfer Buffer
We recommend using 1X NuPAGE ® Transfer Buffer with NuPAGE ®
Antioxidant and 10% methanol for semi-wet transfer of proteins from the
E-PAGE ™ Gel.
Note:
If a large amount of heat is produced during the transfer, use a cooling
unit or perform transfer with cold buffer.
For one blot module, prepare 1000 mL of 1X NuPAGE
®
Transfer Buffer as
follows:
Buffer Component Transfer Buffer with 10% Methano l
NuPAGE ® Transfer Buffer (20X) 50 mL
NuPAGE ® Antioxidant 1 mL
Methanol 100 mL
Deionized Water 849 mL
Preparing
Blotting Pads,
Membrane, and
Gel
1. Use ~ 700 mL 1X NuPAGE
®
Transfer Buffer (see previous page) to soak the
blotting pads of the blot module until saturated. Remove air bubbles by
squeezing the blotting pads while they are submerged in buffer.
Note: Use the blotting pads that fit the XCell II ™ Blot Module. Do not use
the E-PAGE

Blotting Pads for semi-wet transfer.
2. Prepare pre-cut membrane/filter paper sandwiches as described below.
•
PVDF membrane : Pre-wet the PVDF membrane for 30 seconds in
methanol, ethanol, or isopropanol. Briefly rinse in deionized water and
place the membrane in a shallow dish containing 50–100 mL
1X NuPAGE
® Transfer Buffer for several minutes.
•
Nitrocellulose membrane : Place the membrane directly into a tray
containing 1X NuPAGE
®
Transfer Buffer for several minutes.
•
Filter paper : Soak briefly in 1X NuPAGE ® Transfer Buffer before using.
3. Transfer the E-PAGE
™
Gel immediately following electrophoresis. You will
need to cut the gel in half so it can fit into the XCell II

Blot Module. Do not
soak the E-PAGE ™ Gel in transfer buffer.