Continuous Semidry Transfer:

1. Disassemble gel-casting plates so the gel is flat on one side or the other.

2. Using a clean razor, remove the stacking gel and clip the top right corner of the blot (according to the loading).

3. Using gloved hands and tweezers, quickly cut a piece of nitrocellulose to the same size of the gel. (measure the gel area in centimeters)

4. Pre-wet both the gel and nitrocellose in transblot** buffer

5. Cut 16 pieces of Munktell Grade 1F filter paper to the size of the nitrocellulose.

6. Assemble the apparatus as follows:

a. Soak 8 pieces of filter paper in trasblot** buffer and make sure they are air bubble free on the anode plate.

b. Place the pre-wet nitrocellulose filter onto the stack

c. Place the pre-soaked gel onto the nitrocellulose

d. Place 8 more soaked pieces of filter paper on the gel.

e. Purge the stack of any air bubbles with a glass pipette.

f. Assemble the cathode plate and start the transfer

7. The equation to calculate the amperage for a 1 hour transfer is:

a. 0.8 mA / cm² gel.

8. After 1 hour, disassemble apparatus and western blot the membrane.

For discontinuous semidry transfer, see the LKB 2117 Manual for appropriate reagents and assembly conditions.

Transblot** buffer for continuous semidry transfer:

39 mM Glycine 2.93g

48 mM Tris 5.81g

0.03% w/v SDS 0.375 g

20% MeOH 200 ml

QS to 1 liter with DI water.