Western Transfer
Protocol
Instructions are provided below for blotting NuPAGE Gels
using the XCell II Blot Module. For detailed instructions, refer to the NuPAGE
Technical Guide available on our Web site (www.invitrongen)
or contact Technical service.
Transferring One Gel
- Prepare
1 liter of 1x NuPAGE transfer buffer by adding 50 ml 20x NuPAGE transfer
buffer and 100 ml methanol to 800 ml dH2O.
- Soak
blotting pads in 700 ml of 1x NuPAGE transfer buffer.
- Prepare
transfer membranes as recommended by the manufacturer.
- Soak the filter paper briefly in 1x
NuPAGE transfer buffer.
- Place
a piece of pre-soaked filter
paper on top of the gel (adhere to the bottome plate) and remove and
trapped air bubbles.
- Turn
the plate over so the gel and filter paper are facing downwards over a
gloved hand or clean flat surface.
- Place
a pre-soaked transfer
membrane on the gel and remove trapped air bubbles.
- Place
another pre-soaked filter paper on top of the membrane and remove any air
bubbles.
- Place
two soaked blotting pads into the cathode (-) core of the blot module.
- Carefully
pick up the gel/membrane assembly and place on blotting pad in the correct
orientation, so the gel is closest to the cathode core.
- Add
enough pre-soaked blotting pads to rise to 0.5 cm over rim of cathode
core. Place the anode (+) core on top of the pads.
- Hold
the blot module together firmly and slide it into the guide rails on the
Lower Buffer Chamber.
- Insert the Gel Tension Wedge into
the Lower Buffer Chamber and lock the Wedge into position.
- Fill
the blode module with 1x NuPAGE transfer buffer prepared in Step 1 until
the gel/membrane assembly is covered.
- Fill
the Outer Buffer Chamber with 650 ml dH2O.
- Place
the lid on the unit and connect the electrical leads to the power supply.
- Perform transfer for nitrocellulose
or PVDF membranes using 30 V constant for 1 hour. The expected start
current is 220 mA and the end current is 180 mA.
Transferring Two Gels in One Blot Module
- Prepare
1 liter 1x NuPAGE transfer buffer by adding 50 ml 20x NuPAGE transfer
buffer and 200 ml methanol to 750 ml dH2O. Add 1 ml NuPAGE
antioxidant in the buffer for reduced samples.
- Repeat
Steps 2-8 (previous page) twice to prepare 2 gel/membrane sandwiches.
- Place
two pre-soaked blotting pads on the cathode core of the blot module.
- Place
the first gel/membrane assembly on the blotting pad in correct
orientation, so the gel is closest the cathode core.
- Add
another pre-soaked blotting
pad on top of the first membrane assembly.
- Place
the second gel/membrane sandwich on top of the blotting pad in the correct
orientation so the gel is closest the cathode core.
- Proceed
with Steps 11-17 from Transferring One Gel (previous page).
Transfer (hand-written annotations found on the original
copy)
- Rinse
Hyprobe square w/ MeOH, then soak/shake in dH2O.
2.Make 600 ml transfer buffer
(10% MeOH for 1 gel, 20% MeOH for
2): 30 ml 20x, 60 or 120 ml MeOHˆ 600 ml,
reusable.
3. Soak 6-8 foam pads, no bubbles (large square glass container).
4. Take gel apart w/ black spatula tool (over saran wrap).
5. Cut off bottom ~cm and wells.
6. Wet filter paper,
put over gel on plate and transfer gel to filter; transfer to 2 wet filter
if necessary to
straighten.
- Sandwich
the blow module: 2 wet foam pads, wet filter w/ gel face up, wet Hyrobe,
wet pad(s) or if 2 gel, 1 wet foam pad, filter w/ gel, Hyprobe, filter
pads
- Pads
to ~5 cm over.
- Put
on top piece, insert in box so itÕs tight.
* Fill upper chamber with
transfer buffer, lower w/ cold dH2O in cold room.
* 30 V, 180 mA, 1.5-3 hrs