While Running protein gel(s), cut membrane to the size a little larger than gel and wet with methanol for a couple of seconds, then equilibrate membrane with transfer buffer or at least 10min. Also put sponges in transfer buffer (and optionally, put transfer buffer in apparatus on ice)
When electrophoresis is complete, disassemble gel sandwich and remove stacking gel. It is recommended that equilibrate gel in transfer buffer.
In a tray/pan (with buffer filled is recommended but even on bench is accepted), Assemble the transfer system by:
place the transfer cassetee white part on bottom which will be connected to the anode(+).
two layers of prewet sponges.
two layers of filter paper
prewet membrane
gel. (Remember the direction of you loading sequence and the running direction.)
two layers of filter paper, using test tube roll to remove air bubbles among paper, gel and membranes.
two layers of sponge.
Put the ice block in the empty side if using Bio-Rad apparatus.
Close the cassettee and put in the rack in correct orientation (back to black and white to white for Bio-Rad apparatus; and the way you can fill in for Invitrogen one).
Fill tank with transfer buffer until the membrane submerged in the buffer.
Connect to power supply and transfer at 80V(bio-Rad) or 30V(Invitrogen) for 45 min to 2 hours depending on the molecular weight of the protein to be detected.
Turn off the power supply and disassemble the apparatus. Remove membrane from cassetee sandwish and note orientation by cutting a corner or marking with a soft lead pencil. Do not use permenent marker because the ink deffusion will affect immuno reaction.
Keep membrane wet in PBS or TTBS if not for long time storage.
Membranes can be dried and stored in reseable plastic bags at 4oC for 1 year or longer at this point. Prior to further processing, dried PVDF membranes must be placed into a small amount of 100% methanol to wet the membranes, then in distilled water or buffer to remove the methanol.
(optional: The gel (NOT the membrane) can be stained with Coomassie blue to see the efficiency of the proteins transfer)
Immunoblotting
If prestained marker is not used, the membrane can be stained with Pouseau S to visualize the proteins on membrane.
Cut off extra edges of the membrane to fit in the blotting box.
Place membrane in blocking buffer and shake/roll at room temperature for 1 hour.
Dilute primary antibody at the manufacturer suggested dilution rate (usually 500-2000) in incubation buffer and incubate membrane at room temperature for 1 hour or at 4oC over night with shaking.
Wash membrane with washing buffer 3 times at 10-15min each.
Incubate membrane in secondary antibody solution (1:5000 is commonly used dilution) at room temperature for 1 hour.
Rinse membrane briefly and wash membrane 3 times at 10-15min. preferably wash one more time with the buffer without detergent will favor the signal duration.
Visualization of target protein
Mix reagent A and B from ECL or Supersignal Kit at 1:1.
Drain washing buffer from membrane and add the mixture. Agitate for 1-5min.
Remove membrane, drain and place on a sheet of plastic wrap. Fold wrap back onto membrane to form a liquid-tight enclosure, make sure to remove bubbles and extra liquid.
In a dark room, place membrane face to film and expose for 1 min as a start. Develop film in Konica SRX-101A developing machine. Adjust exposure time to get a series of different darkness films.
Membranes can be washed with PBS and store for reprobing.