C.B.S. Scientific EBU-102P - Manual (Page 8)

C.B.S. œ Scientific
8
Electro-Blotting System
SECTION 3
Instructions for Use
3.1
Blotting Unit Preparation
1. Place the blotting chamber on a level work surface in an authorized work area.
2. Consult applications table 4.1 for type of transfer, buffer system, membrane type and power
settings.
Coolant Circulation:
For coolant circulation, connect a heavy wall tubing which will not kink (hose clamps are
optional), to the tubing adapters supplied with the unit (excessive pressure will damage the seal
between the glass and the base and possibly create leaks). Attach a regulated pump or
recirculating water bath (follow manufacturer’s instructions), recommended flow is 500 ml per
minute or 15psi. Do NOT use tap or house water as it can be subject to large fluctuations in
pressure.
1. Place, do NOT drop, a magnetic spin bar (not supplied by C.B.S.) in the bottom of the tank.
This maintains pH, buffer and heat circulation during the electroblot.
2. Place tank on top of the magnetic stirrer.
3. Connect to a cold water supply (see coolant circulation above).
4. Buffer should be pre-cooled to 10
°C.
3.2 Preparing the Gel for Transfer
The EBU-202 can hold up to 4 gel cassettes simultaneously with space provided for a magnetic
stir bar to allow buffer circulation and heat exchange for uniform transfers. The EBU-102 holds
up to 3 gel cassettes simultaneously. When the resolving gel electrophoresis is complete,
proceed with staining and photo-documentation if applicable. Cut one corner off the gel so that
correct orientation is maintained throughout the procedure.
Preparation of Electroblotting Components
1. Cut transfer membrane to size of gel or sufficient dimensions to cover the relevant
bands. Also, pre-cut Whatman-type 3MM filter paper to same size and soak in
transfer buffer until completely saturated (15 to 30 minutes).
2. Pre-wet membrane chosen according to manufacturer’s instructions. Nylon or Nylon-
supported nitrocellulose should be soaked in ddH
2
O. PVDF in MeOH. Equilibrate all
types in transfer buffer for 3 minutes.
3. Pre-equilibrate gel in transfer buffer to be used prior to electroblotting for 10 to 30
minutes depending on gel thickness.
4. Open the transfer cassette and submerge the red (+) panel in a shallow plastic or
glass (Pyrex) dish. Fill with enough buffer to cover entire cassette. Submerge a
single Scotch Brite
® pad and gently tease to release trapped air bubbles until pad is
completely saturated.
5. Layer on top of the Scotch Brite pad your transfer membrane. (Some prefer to layer
1 to 2 sheets of saturated 3mm filter paper). Carefully search for and remove
trapped air bubbles at each layer by rolling a pipet over the surface.
Note: Scotch Brite pads will degrade/compress over time. To maintain tightness
in the stack, add filter paper or replace pads.
6. Apply gel correctly oriented to submerged transfer membrane. Check for air pockets
and remove. Note: Some proteins will begin transfer immediately upon contracting