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Appendix : Trouble shooting 1 General aspects ? Quality of chemicals should be at least of analytical grade (p.a.) ? Double-distilled or deionized (Millipore ) water (conductivity < 2 μS) should be used ? Urea and acrylamide/bisacrylamide solutions should be prepared freshly ? Deionize urea prior to use ? Do not heat urea-containing buffers > 37°C; otherwise protein carbamylation may occur ? Filter all solutions. Use clean and dust-free vessels

2 Sample preparation ? Sample extraction buffer (Lysis buffer) has to be prepared freshly. Alternatively, make small portions (1 ml) and store frozen in eppendorf vials at -70°C. Lysis buffer thawn once should not be refrozen again! ? Add protease inhibitors during Cell lysis if necessary. Note: several protease inhibitors are inactivated by DTT and/or mercaptoethanol! ? To remove insoluble material, the protein extract should be spun for 1 h at 40,000 g

3 Gel casting ? Ammonium persulfate solution should be prepared freshly. A 40% solution of ammonium persulfate may be used for 2-3 days if stored in a refrigerator, whereas less concentrated solutions should be prepared the day you use them ? TEMED should be stored under nitrogen and replaced every six months ? The glass plate which bears the U-shaped frame should be treated with RepelSilane to avoid sticking of the gel to the glass plate after polymerization ? Glycerol (37.5%) is incorporated into the stacking gel of horizontal SDS gels in order to diminish electroendosmotic effects ? If SDS gels are cast onto GelBond PAGfilm, GelBondPAGfilm should be washed 6 x 10 min prior to use to avoid "spot streaking" upon silver-staining ? For proper polymerization, acidic as well as basic Immobiline starter solutions should be titrated to pH 7 with NaOH and HCl, respectively, prior to IPG gel casting ? After polymerization, IPG gels have to be washed thoroughly (6 x 10 min) with deionized water to remove buffer ions and any unpolymerized material ? Washed IPG gels are impregnated with glycerol (2%) for 30 minutes and dried overnight at room temperature in a dust-free cabinet with the help of a fan ? The surface of the dried IPG gels has to be covered with a sheet of plastic film prior to storage at -20°C

Observed problems: Gel did not polymerize properly

4 Reswelling of IPG strips ? Prior to IEF, IPG dry gels have to be cut into individual IPG strips with the help of a paper cutter. During cutting, the surface of the IPG strips has to be protected by a sheet of plastic film to avoid damage of the gel surface ? IPG strips have to be rehydrated to their original thickness of 0.5 mm ? IPG gel reswelling time depends on the composition of the rehydration buffer. If the rehydration solution contains high concentrations of urea (> 8 M) and detergents (>1%), rehydration should be performed for 6 hr at least or, better, overnight