Preparation of semi-dry transfer buffer
Preparation of Semi-dry Transfer Buffer
- Cut the transfer membrane and four pieces of Whatmann 3 MM paper to gel size (7 x 8 cm).
- 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.
- Wet the porous pads as well as the four pieces of Whatmann 3 MM paper with transfer buffer.
- Remove the gel from the cassette and equilibrate the gel for 5 minutes in transfer buffer.
- Mount the transfer sandwich analogue to the tank blot sandwich and place it into the semi-dry blotter.
- 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.
|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|
- 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