• for each strip place 350 µL of rehydration soln into reswelling tray. 
• remove protective covering from IPG strip
• lay strip, gel side down, on rehydration soln
• overlay each strip with 2ml of rehydration cover fluid (light mineral oil).
• allow strips to rehydrate at r.t. for at least 12h!

Urea Stock
8M Urea
2% CHAPS
aliquot and freeze.

Rehydration soln
urea stock +
2.8 mg/ml DTT
48.6µL/ml IPG buffer (pH 3-10)

Day 2 Sample Preparation & first dimension -- IEF

• Take IP pellet and resuspend in 200 µL IEF sample buffer
• Incubate for 2-4 hours at r.t.
• Allow beads to settle - transfer supernatant to new tube
• Mix with 140 µL rehydration solution

• Turn circulating water bath on and set to 20°C -  connect to ceramic base of the Multiphore II apparatus.
• Pore ~5 ml mineral oil on the ceramic plate
• Place the drystrip/electroholder on top of mineral oil
• Pour 5ml of light mineral oild on the drystrip/electrode holder.
• Place a drystrip aligning tray, with the grooves facing up, on top of the mineral oil.
• Cut 2 IEF electrode strips 110mm long + 2 IEF electrode strips 10mm long for each drystrip to be run. Wet with distilled water -- remove excess water
• rinse each drystrip briefly with water

• Lay each dry strip in a groove in the aligning tray, gel side up.
• Pointed end of drystrip should pont to black/anode (+) of apparatus
• Place on short IEF electrode strip parallel to, and sligthly overlapping the gel
• Place long IEF electrode strip perpendicular to drystrip,on top of the short IEF/gel strip
• Line up electrodes with long IEF strips and clip them on the drystrip tray/holder
• Fill the tray/holder with drystrip coverfluid (light mineral oil) to completely cover the drystrips and electrode strips (about 50-60 mL).
• Attach cover to apparatus
• Attach to EPS 3501XL power supply - you are ready to run!!!!

Turn off "current check" and program run

The dye will migrate to the anode.

Day 3 Second Dimension -- PAGE

• Disassemble the IEF apparatus -- remove drystrips -- rinse briefly in water
• Incubate strips with reducing solution for 15 minutes (use a 100ml graduated cylinder with rocking)
• Incubate strips in alkalating solution (optional simple experiments but probably necessary for MALDI analysis) for 15 minutes - with rocking
• remove strip -- trim excess backing away -- note strip orientation

Reducing solution
SDS equilibration buffer
+ 10 mg/ml DTT

Alkylating solution
SDS equilibration buffer
+ 25 mg/ml iodoacetamide

SDS equilibration buffer
50ml 1.5mM Tris pH 8.8
6M Urea
30% glycerol
2% SDS
Bromophenol Blue

Place strip on top of Comb-less SDS-PAGE gel

• For MALDI --go to MALDI page