Preparation of semi-dry transfer buffer

Preparation of Semi-dry Transfer Buffer

  1. Cut the transfer membrane and four pieces of Whatmann 3 MM paper to gel size (7 x 8 cm).

  2. Equilibrate the membrane in transfer buffer. By use of PVDF membranes equilibrate the membrane first for 2 minutes in methanol and then for additional 5 minutes in transfer buffer.

  3. Wet the porous pads as well as the four pieces of Whatmann 3 MM paper with transfer buffer.

  4. Remove the gel from the cassette and equilibrate the gel for 5 minutes in transfer buffer.

  5. Mount the transfer sandwich analogue to the tank blot sandwich and place it into the semi-dry blotter.

  6. Transfer is done at room temperature with 0.8 - 1.5 mA/cm2 gel area for approx. 1 hour. 

By blotting of proteins of differently large sizes the use of a discontinuous blotting buffer system is recommended.


Transfer buffer:

Anode buffer 1: 300 mM Tris, pH 9.5
Anode buffer 2: 30 mM Tris, pH 9.5
Cathode buffer: 40 mM 6-Aminohexanoic acid, 20 % (v/v) methanol


Blotting sandwich:

Anode (+)

  • 2 filter papers moisted with anode buffer 1
  • 2 filter papers moisted with anode buffer 2
  • Transfer membrane
  • 3 filter papers moisted with cathode buffer

Cathode (-)

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