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Acrylamide gels
Prepare the monomer solution and pour the gel
Prepare the required amount of monomer solution. Deaerate and
add the initiator and catalyst just prior to pouring the gel. Pipette the
solution into one corner of the sandwich, taking care not to intro-
duce any air bubbles. See below for the appropriate solution level
according to the application.
No stacking gel (Continuous system) Fill solution to just below
the top of the upper plate edge. If bubbles are trapped, remove with
a pipette or syringe. Introduce a comb (at a slight angle) into each
sandwich, taking care not to trap air bubbles under the teeth.
Club sandwich Pipette the solution into both sandwiches, filling
each to the same level below the notched edge.
Stacking gel Fill solution to 3–4 cm below the top of the glass
plate. This height allows 1 cm of stacking gel below the wells.
Pour the gel and apply an overlay (see step 2). After the gel is set,
prepare the stacking gel as described below.
2-D electrophoresis (Discontinuous protein system) Fill monomer
solution to about 1 cm below the top of the glass plate to allow 4–5
mm for the IPG strip or tube gel and an agarose seal. (A stacking gel
will require extra space). Seal the IPG strip or tube gel in place with
agarose dissolved in running buffer. Take care to avoid trapping any
air bubbles between the first- and second-dimension gels.
Overlay each gel with a thin layer of water-saturated butanol, water,
or diluted gel buffer to prevent gel exposure to oxygen. Slowly
deliver the overlay solution from a glass syringe fitted with a 22-
gauge needle. Apply the solution near the spacer at one side of the
sandwich and allow it to flow across the surface unaided.
Allow the gel to polymerize for a minimum of 1 h.
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p12
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