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
