Blotting

 

Membranes:       Nitrocellulose,

                           Nylon,

                           PVDF,..

 

                           Pore size : 0.1 - 0.45 µm

 

Transfer:        

- via electric charge: electro-blotting

·    Tank-Blotting

·    Semidry-Blotting

 

- via buffer

 

Binding to the membrane :        hydrophobic Interaction

 

Protocol for electro-blotting:

 

1.       Cut membrane to gel size

2.       Wash membrane with methanol (2s) and water (2 min)

3.        Equilibrate membrane in CAPS - buffer (10 mM CAPS, 10 % Methanol pH = 11.0) (> 15 min)

           (CAPS = Cyclohexylamino-1-propansulfonsäure)

4.       Subsequent to electrophoresis wash gel 5 min with transfer buffer

5.       Put membrane on gel, add 2 layers of Whatman 3MM paper and mount in Blotting-apparatus

6.       Transfer of 10 kD - protein, 0.5 mm gel at 0.5 A in about10 minutes, larger proteins up to 1 h

7.       Long transfer time increases yield of large proteins but decreases yield of small ones

8.       Subsequently wash membrane 2-3 times with aqua bidest.