Western Blotting

Run SDS-PAGE as described in SDS-PAGE protocol

  1. Once SDS-PAGE is finished, trim off wells and stacking gel with a razor blade, cut the bottom left corner of the gel
  2. Cut a piece of pvdf to the size of the gel to be transferred and cut the bottom left corner also. Pre-soak the pvdf* in 100% methanol for at least 5 minutes prior to use.
  3. Pre-equilibrate (per gel) 2 sheets of thick filter paper, 2 coarse wafers and the gel to be transferred, in running buffer (TGS)
  4. Assemble the transfer “sandwich” as follows: hold the sandwich case in the palm of your hand with the clear plastic in your palm and the black plastic at a 90 degree angle. Layer 1 wafer, 1 sheet of filter paper, the pvdf, lay the gel carefully on top of the pvdf making sure there are no bubbles, then add the second filter paper and the second wafer. Close the sandwich case and put in the blotting apparatus with the black side of the case facing the black side of the apparatus.
  5. Place the ice-block in the apparatus (the ice-block should be rinsed, refilled with milliQ water and returned to the -20 C freezer after each use)
  6. Fill the tank up to mid-way through the top layer of holes in the sandwich case with TGS buffer
  7. Set the power pack to run at 250mA constant for 1 hour
  8. After the one hour period disassemble the sandwich case. If prestained molecular weight markers were used, these should be visible on the pvdf, otherwise make sure that the cut portion of the membrane is still on the bottom left.
  9. Rinse the pvdf carefully with milliQ water and add blocking buffer. Block overnight at 4 C or for several hours at room temperature
  1. Prepare primary antibody in blocking buffer (~10ml will suffice). Add primary antibody to membrane (first discarding blocking buffer). Incubate at room temperature on orbital shaker for approximately 2 hours (this may change depending on antibody)
  2. Discard the primary antibody and wash the membrane three times for 10 minutes each time with TBS-T
  3. Prepare the secondary antibody (AP-conjugate) in blocking buffer. Add to the membrane and incubate at room temperature for 1 hour
  4. Discard the secondary antibody and wash the membrane three times for 10 minutes each time with TBS-T
  5. Add the alkaline phosphatase substrate (BCIP/NBT from Sigma) - just enough to cover the membrane, ~ 5ml will suffice.
  6. Wash the membrane with water once the signal has developed sufficiently
  7. Dry membrane on a piece of kimwipe

** *nitrocellulose may also be used

10X TBS

  • 24.2g Tris base
  • 80g NaCl
  • pH 7.6, make up to 1L with MilliQ water

TBS-T

  • 50ml 10X TBS
  • 0.5ml Tween 20
  • 450ml MilliQ water

Blocking buffer

  • 5g of Non fat dried milk
  • 100ml of TBS-T

10X running buffer (TG)

  • 30.3 g Tris base
  • 144.1 g glycine
  • water to 1 liter

Running buffer (TG)

  • 100 ml 10X stock
  • 500 ml H2O
  • 200 ml methanol
  • water to 1 L