Western Blotting - wet (submersed) method

Notes

Blotting

  1. Take the gel(s) out of the running apparatus and cut a small triangle off the bottom right of the gel as you look at it. Trim the gel(s) to remove wells and the sticky-uppy bit at the bottom.
  2. Equilibrate gels in Western Transfer (Towbin's) Buffer (WTB) at 4°C for 15 min.
  3. Cut 2 pieces of Whatman 3MM or equivalent per gel, allowing for a ~2 cm border, and soak in WTB. Place two Scotch pads per gel into WTB, all in the coldroom.
  4. Cut one piece of PVDF (Millipore Immobilon™-P) membrane, 0.5 cm larger than the gel. Wet the membrane in methanol.
  5. Place the gel on one piece of 3MM, resting on a wet Scotch pad, with the cut triangle on the bottom left. Roll out the bubbles with a pipette. Trim the 3MM to fit the gel (you'll need sharp scissors). Place the PVDF membrane on top of the gel, followed by the second piece of 3MM. Roll out the bubbles with a pipette. Trim the PVDF and second piece of 3MM to fit. Place the second Scotch pad on top.
  6. Arrange the entire sandwich in one of the black and grey hinged container thingies, so that the gel side is towards the black side and the membrane towards the grey (you might want to construct the sandwich on one of these thingies). If you've done this right, the cut edge will be on the left as it lies on the black side. Close up the hinged thingy and ensure that the clamps engage.
  7. Pour cold WTB into the blotting cell to the first fill line. Insert the sandwich so that the black side is towards the cathode (BLACK electrode). Top up with WTB.
  8. Fit the lid, and blot at 200 mA for 1 hour, in the coldroom. Timing depends on the transferability of your protein - large (> 150 kDa) proteins take longer but 1 hour/200 mA should be enough for most of what we do. Double the time and/or the current if it isn't.

Blocking

  1. Disconnect and disassemble the blotting rig. Rinse all non-disposable parts with dH2O (tris/glycine leaves nasty marks when the methanol evaporates).
  2. Peel apart the blotting sandwich. Dump the gel in Coomassie to check for transfer. If you used prestained markers you should be able to see these on the PVDF.
  3. Block the membrane in 3% dried milk powder, 0.02% Tween 20 either at room temperature for 1 hour or at 4°C overnight. Add azide if blocking overnight.
  4. Wash the membrane 3 x 5 minutes with shaking in PBS-T.

At this stage the membrane can be left wet, wrapped in Saran Wrap, in the fridge for days/weeks.

Probing

  1. Incubate membrane in ~ 10 ml primary antibody diluted in antibody diluent for 45 - 60 minutes at room temperature.
  2. Wash 3 x 5 minutes in PBS-T.
  3. Incubate membrane in ~ 10 ml secondary antibody (HRP-conjugate) diluted in antibody diluent for 20 - 30 minutes at room temperature.
  4. Wash 3 x 5 minutes in PBS-T.

Detection

  1. We have an 'ECL' kit for this. RTFM.

Buffers

WTB 10x WTB PBS-T Ab diluent
25 mM Tris
192 mM glycine
20% methanol 		30 g Tris base
144 g glycine
per litre 		PBS
0.1% Tween 20
 		 PBS
0.05% BSA
 

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