C.B.S. Scientific EBU-222 - Manual (Page 7)

EBU-222 Instruction Manual, version 7/28/2011 7 www.cbsscientific.com
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-222 can hold up to 2 gel cassettes simultaneously with space provided for a magnetic stir bar to allow
buffer circulation and heat exchange for uniform transfers. 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 nitrocellu-
lose should be soaked in ddH2O. 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 your transfer membrane on top of your sponge pad. (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.
6. Apply gel correctly oriented to submerged transfer membrane. Check for air pockets and remove. Note:
Some proteins will begin transfer immediately upon contracting the transfer membrane. Disturbing or mov-
ing the gel/membrane interface can result in a smeared blot.
7. Quickly follow with 1-2 sheets of saturated 3mm filter paper (optional).
8. Place second saturated sponge pad over sandwich assembly and latch the cassette into the closed
position.