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

  1. Prepare 1 liter of 1x NuPAGE transfer buffer by adding 50 ml 20x NuPAGE transfer buffer and 100 ml methanol to 800 ml dH2O.

 

  1. Soak blotting pads in 700 ml of 1x NuPAGE transfer buffer.

 

  1. Prepare transfer membranes as recommended by the manufacturer.

 

  1.  Soak the filter paper briefly in 1x NuPAGE transfer buffer.

 

  1. Place a piece of  pre-soaked filter paper on top of the gel (adhere to the bottome plate) and remove and trapped air bubbles.

 

  1. Turn the plate over so the gel and filter paper are facing downwards over a gloved hand or clean flat surface.

 

  1. Place a pre-soaked  transfer membrane on the gel and remove trapped air bubbles.

 

  1. Place another pre-soaked filter paper on top of the membrane and remove any air bubbles.

 

  1. Place two soaked blotting pads into the cathode (-) core of the blot module.

 

  1. 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.

 

  1. 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.

 

  1. Hold the blot module together firmly and slide it into the guide rails on the Lower Buffer Chamber.

 

  1.  Insert the Gel Tension Wedge into the Lower Buffer Chamber and lock the Wedge into position.

 

  1. Fill the blode module with 1x NuPAGE transfer buffer prepared in Step 1 until the gel/membrane assembly is covered.

 

  1. Fill the Outer Buffer Chamber with 650 ml dH2O.

 

  1. Place the lid on the unit and connect the electrical leads to the power supply.

 

  1.  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

 

  1. 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.

 

  1. Repeat Steps 2-8 (previous page) twice to prepare 2 gel/membrane sandwiches.

 

  1. Place two pre-soaked blotting pads on the cathode core of the blot module.

 

  1. Place the first gel/membrane assembly on the blotting pad in correct orientation, so the gel is closest the cathode core.

 

  1. Add another  pre-soaked blotting pad on top of the first membrane assembly.

 

  1. Place the second gel/membrane sandwich on top of the blotting pad in the correct orientation so the gel is closest the cathode core.

 

  1. Proceed with Steps 11-17 from Transferring One Gel (previous page).

 

Transfer (hand-written annotations found on the original copy)

 

  1. 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.

 

  1. 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

 

  1. Pads to ~5 cm over.

 

  1. 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