2) Remove gel from plates, do not fix, and stain for approximately 10 min.
3) Destain gel.
4) Rehydrate gel in water for 5-10 minutes.
5) Cut out region of gel containing band of interest and mince gel slice into ‰1mm cubes. This can be done conveniently in a weigh boat.
6) Prepare elution; cup by securing a single layer of dialysis membrane beneath each of the wells with the retaining rings. Place a plastic mesh screen over the collection (smaller) well.
7) Prepare buffer chamber by placing a piece of Whatman 3mm paper in the divider
slots of each electrode chamber and fill all chambers with 1x Laemmli gel running
buffer, containing SDS, to approximately 1cm below the top of the central
divider/cup holder. Buffer must not rise over this divider when the elution cups are
placed on it. The paper dividers prevent bubbles generated at electrodes during
elution from collecting under the wells and preventing current flow.
If a very small amount of protein is being eluted (in the pg range) such as might be
recovered from a biologically significant source, not a fusion protein, it may be useful to
add protease inhibitors to the elution buffer. For example, add PMSF to 1mM and
aprotinin to 1/1000.
8) Pile minced gel pieces into sample (larger) well. Be careful not to trap air in the
bottom of the wells or create air pockets in the mass of minced gel.
9) Place elution cups in the chamber straddling the center divider. Be certain not to trap air bubbles underneath the wells.
10) Elute in cold-room at 150 V, constant V, for ‰12 hrs. Be certain that there is current
flowing through the chamber. The Coomassie Blue dye will collect in the collection
chamber and provide an indicator of completion of elution.
If no current flow is detected the most likely cause is a bubble either in or under one of the
wells. It can sometimes be difficult to see these bubbles so inspect closely, removing the gel
slices if necessary.
11) When elution is complete, remove buffer from elution cup with pasteur pipette,
remove gel slices from collection well side of cup and remove remaining buffer down
to rim of the collection well.
12) Remove screen and recover eluted protein from collection well with Pipetman or equivalent. The volume of the collection well is 200µl. The collection well can be washed by pipetting solution up and down, gently so as to prevent foaming, or with fresh buffer.
13) Dialyse the sample as necessary.
14) Clean the apparatus thoroughly.