Hoefer IEF100 - Manual (Page 61)

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3. SDS Equilibration Buffer Solution
This solution is used after IEF, and before second dimension PAGE. The IPG
strips are immersed in excess solution to raise the pH of the strip buffer so
that it is suitable for PAGE, and to coat the proteins in SDS uniformly so
that they migrate properly in the second dimension gel.
Prepares 200 ml
6 M urea, 75 mM Tris-HCl pH 8.8, 29.3% glycerol, 2% SDS, 0.002%
bromophenol blue
Final Concentration Amount
Urea (FW 60.06) 6 M 72.1 g
1.5M Tris-HCl, pH 8.8 stock solution 75 mM 10.0 ml
Glycerol (87% w/w) 29.3% (v/v) 69 ml
SDS (FW 288.38) 2% (w/v) 4.0 g
Bromophenol blue 0.002% (w/v) 4 mg
Deionized water to 200 ml
Aliquot into 30 ml aliquots and store frozen at -20 °C or below.
24 cm IPG’s require 5 –10 ml per strip per equilibration step. Shorter strips
can use proportionately less volume per equilibration step.
Equilibration Procedure
1 Thaw two aliquots of the equilibration solution.
2 Add 10 mg/ml DTT to one solution.
3 Place the IPG strips in the rehydration/equilibration tray.
4 Add 6.5 ml of solution to each slot containing an IPG strip.
5 Place on rocker for 10 –15 minutes.
After equilibration, discard the first equilibration solution in an
appropriate manner.
6 Add 25 mg/ml Iodoacetamide (IAA) to the second aliquot of
equilibration solution.
7 Add 6.5 ml of solution to each slot containing an IPG strip.
8 Place on rocker for 10 –15 minutes.
After equilibration, discard the second equilibration solution in an
appropriate manner.
Following equilibration, the IPG strips are placed on the top of the second
dimension gel, and sealed into place with the agarose overlay.
Note: IPG strips should be
equilibrated just prior to second
dimensin PAGE. Do not equilibrate
the IPG strips before storing at
-20 °C.