WET WESTERN BLOT THERMOFISHER
For Blotting NuPAGE ® Novex ® Bis-Tris or Tris-Acetate Gels: Prepare 1,000 mL of 1X NuPAGE ®
Transfer Buffer using the NuPAGE ® Transfer Buffer (20X) as follows:
NuPAGE Transfer Buffer with 10% methanol provides optimal transfer of a single gel in the blot module. If
you are transferring 2 gels in the blot module, increase the methanol content to 20% to ensure efficient
transfer of both gels.
Preparing Blotting Pads
Use ~ 700 mL of transfer buffer to soak the blotting pads until saturated. Remove air bubbles by
squeezing the blotting pads while they are submerged in buffer. Removing air bubbles is essential as they
can block the transfer of biomolecules.
Preparing Transfer Membrane and Filter Paper
PVDF membrane: Pre-wet the PVDF membrane for 30 seconds in methanol, ethanol, or isopropanol.
Briefly rinse in deionized water and then place the membrane in a shallow dish containing 50–100 mL
transfer buffer for several minutes.
Filter paper: Soak briefly in transfer buffer immediately before using.
Gel: Use the gel immediately following the run. Do not soak the gel in transfer buffer.
Transferring One Gel
1. Wet the surface of the gel (step 8, above) with the transfer buffer and place pre-soaked transfer
membrane on the gel. Remove air bubbles by rolling a glass pipette over the membrane surface.
2. Place the pre-soaked filter paper on top of the transfer
membrane. Remove any trapped air bubbles.
3. Place 2 soaked blotting pads into the cathode (–) core of the blot module. The cathode core is the
deeper of the 2 cores and the corresponding electrode plate is a darker shade of gray. Carefully pick up the
gel membrane assembly with your gloved hand and place on the pad in the same sequence, such that the
gel is closest to the cathode plate.
4. Add enough pre-soaked blotting pads to rise 0.5 cm over the rim of the cathode core. Place the anode
(+) core on top of the pads. The gel/membrane sandwich should be held securely between the two halves
of the blot module ensuring complete contact of all components.
Note: To ensure a snug fit, use an additional pad since pads lose their resiliency after many uses. Replace
pads when they begin to lose resiliency and are discolored.
5. Position the gel membrane sandwich and blotting pads in the cathode core of the XCell II ™ Blot Module
to fit horizontally across the bottom of the unit. There should be a gap of ~ 1 cm at the top of the electrodes
when the pads and assembly are in place.
6. Hold the blot module together firmly and slide it into the
guide rails on the lower buffer chamber. The blot module fits into the unit in only one way, such that the (+)
sign is seen in the upper left-hand corner of the blot module. The inverted gold post on the right-hand side
of the blot module fits into the hole next to the upright gold post on the right side of the lower buffer
chamber.
7. Depending on the mini-cell that you are using, follow the appropriate instructions for positioning the
wedge:
For XCell SureLock ™ Mini-Cell, place the gel tension wedge such that the vertical face of the wedge is
against the blot module. Push the lever forward to lock it into place.
For XCell II ™ Mini-Cell, place the front wedge (without the screw hole) such that the vertical face of the
wedge is against the blot module. Slide in the rear wedge and push it down firmly.
Note: When properly placed, the rear wedge will not be flush with the top of the lower buffer chamber.
There will be a gap between the rear wedge and lower chamber.
8. Fill the blot module with transfer buffer until the gel/membrane sandwich is covered in transfer buffer. Do
not fill all the way to the top as this will generate extra conductivity and heat.
9. Fill the outer buffer chamber with ~ 650 mL deionized water by pouring in the gap between the front of
the blot module and front of the lower buffer chamber. The water level should reach approximately 2 cm
from the top of the lower buffer chamber. This serves to dissipate heat produced during the run.
Note: If you accidentally fill the outer buffer chamber with the transfer buffer, it will not adversely affect the
transfer. The liquid in the outer buffer chamber serves as a coolant. We recommend adding deionized
water to the outer buffer chamber to avoid any exposure of the mini-cell to methanol as the mini-cell is
susceptible to ethanol.
10. Place the lid on top of the unit.
