• Rinse and blot the rehydrated IPG gel strips to remove excess rehydration solution (not to be done with the IPGphor); otherwise urea crystallization on the surface of the IPG gel strips might occur and disturb IEF patterns
• If IPG strips are rehydrated in the IPG reswelling tray or in IPGphor strip holders, avoid trapping air bubbles between the IPG strip and the bottom of the tray or the strip holder Distribute the sample solution evenly beneath the IPG gel strip. Cover the IPG strips with a layer of silicone oil during reswelling to prevent evaporation of the reswelling buffer
5 First dimension (IEF-IPG)
5.1 Application of rehydrated IPG strips onto the cooling plate of the electrophoresis chamber
• Use kerosene exclusively to facilitate contact between IPG strips and cooling block
• Use distilled water as electrode solution exclusively
• Make sure that the orientation of the IPG gel strips on the cooling block of the IEF chamber is correct (acidic end facing towards anode)
5.2 Sample application
• Sample may be applied by in-gel rehydration or by cup-loading
5.2.1 Sample application by cup-loading
• When sample is applied into cups, do not apply less than 20 μl of sample solution
• Samples may be applied near anode or cathode. In the case of unknown samples it should be checked which sample application area provides better results
• Sample solution should not be too concentrated (max. 10 mg protein/ml) to avoid protein precipitation at the sample application point. If you are in doubt, better dilute the sample with Lysis buffer and apply a larger volume instead
• Sample solution shoud not contain too high concentrations of salt. Either desalt, or dilute with lysis buffer and apply a larger volume instead. Apply low voltage for slow sample entry.
5.2.2 Sample application by in-gel rehydration
• When using the reswelling tray for in-gel rehydration, the sample volume has to be limited to the size of the IPG strip so that no superfluous sample solution is left in the tray. For a 180 mm long and 3 mm wide IPG strip, the correct sample volume is about 350 μl. When the reswelling tray is used for sample application, one should be aware that
high molecular weight, alkaline and/or membrane proteins may not enter the IPG gel matrix properly
5.3 Isoelectric focusing
• Never pre-focus IPG strips; otherwise poor sample entry occurs due to the very low conductivity of the gels
• Electrode strips should be humid, but not too wet. Remove superfluous liquid by blotting with filter paper
• Keep temperature of the cooling block at 20°C
- For better sample entry, start IEF with a low voltage gradient (150 V for 30 min, followed by 300 V for 60 min). For micropreparative runs (cup loading) with a high sample volume (100 μl) start IEF at low voltage for several hours (200 V for 5-6 hours), followed by 1500 V overnight, before voltage is raised to 3500 V
• IPGphor: For improved sampe entry apply low voltage (30 V) during rehydration. Then
raise voltage gradually (200 V, 500 V, 1000 V for 1 h each) and continue with max. 8000 V up to the
steady state
• Focusing time depends on gel length, pH-gradient and gel additives (carrier ampholytes etc.). Focusing time is shorter when separation distance is shorter, or when wide-range pH-gradients are used, or when carrier ampholytes are added to the reswelling solution
• When running very basic and/or narrow-range IPGs, cover the IPG strips with a layer of degassed silicone oil flushed with argon
• After completion of IEF, IPG strips should be stored frozen at -78°C (unless immediately used for the second dimension)
Observed problems:
IPG gel strips "burn" near the electrode (strips)
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