Troubleshooting first-dimension IEF: Ettan™ IPGphor™ 3 Isoelectric Focusing System

Table 20 lists possible problems that might be encountered during IEF and how to solve them, and Table 21 lists problems and solutions when using Ettan Manifold.

Table 20. Troubleshooting first-dimension IEF: Ettan™ IPGphor™ 3 Isoelectric Focusing System.

Symptom	Possible solutions
Problems indicated by LCD messages
Lid open step 1, close to continue	The safety lid is not properly closed. When the safety lid is open, the system has an automatic voltage cutoff safety feature. In order for the protocol to proceed, the safety lid must be closed.
Locked screen in edit mode	Turn off the mains power switch to reset the instrument.
Blank display	If no electrical components are functioning (e.g. HV lamp does not light and the cooling fans are motionless), check the fuses in the mains power module.
Diagnostic program indicates component failure	Note the component that failed and press the START key to continue through the diagnostic program. Call your local Cytiva sales office for further information on how to remedy the failure.
Power delivery
Current too low or zero	At least two of three pressure pads on the lid adaptor of IPGpho™r 3 under the safety lid should press gently against the strip holders to ensure electrical continuity between the strip holder electrodes and the electrode areas on the platform. The gel must be evenly and completely rehydrated to conduct current. Make sure the proper amount of rehydration solution is applied to the IPG strip holder and allow a minimum of 10 hours for rehydration.
Voltage limit not reached	The ionic strength of the rehydration solution is too high; reduce the IPG buffer concentration; use a mixed-bed ion-exchange resin to remove ionic breakdown products of urea or other additives. Desalt the sample or prepare the sample so that the salt concentration is less than 10 mM.
Sparks or burning in strips	Reduce the current limit. Do not exceed 50 µA per strip. Prevent the IPG strip from drying out by always applying Immobiline® DryStrip Cover Fluid immediately after strip placement in rehydration buffer. Ensure that the IPG strip is fully rehydrated along the entire length of the strip. The IPG strip should be in complete contact with the correct volume of rehydration solution. Remove any air bubbles trapped under the IPG strip. Desalt the sample or prepare the sample so that the salt concentration is less than 10 mM. De-ionize additives to the rehydration solution. Excessive charged material in the sample or rehydration buffer can lead to electroendosmosis, which could dry out part of the strip, possibly leading to arcing and burning in this region.

Table 21. Troubleshooting first-dimension IEF: Employing the Manifold.

Symptom	Possible cause	Remedy
Current is too low or zero	Electrical continuity is impeded.	Check the external electrode contacts: Ensure correct placement of the electrode assemblies such that there is metal-to-metal contact with the appropriate electrode contact area. Check the internal electrode contacts: The gel (which becomes visible because of the dye in the rehydration solution) must contact both electrodes in the Manifold through the paper wicks and/or paper bridge parts. Check that the IPG strip is fully rehydrated along its entire length. Electrical contact at the electrodes is reduced by incomplete rehydration. Check that the paper wicks are present and properly positioned.
Voltage too low or does not reach maximum set value	Ettan™ IPGphor™ protocol settings are incorrect for the experiment.	Check that the current limit is properly set. Check that the actual number of Immobiline® DryStrip gels on the Ettan™ IPGphor™ platform is the same as the number of gels entered in the protocol.
	Conductivity/ionic strength is too high.	Prepare the sample to yield a salt concentration less than 10 mM. The recommended IPG Buffer concentration is 0.5%. A maximum of 2% is advisable only if sample solubility is a problem. High conductivity can also arise from the use of poor quality urea or other denaturants. Urea is also prone to decomposing to charged breakdown products. Higher conductivity salts and ionic impurities in the sample can raise the conductivity of the strip. Shorter length IPG strips (e.g. 7 cm strips) will not reach 8000 V. The distance between the electrodes is shorter so that the voltage gradient (V/cm) required to reach the 50 µA current limit is reached at a lower overall voltage.
Sample leaks from cup	Incorrect cup placement.	Check that the feet of the cups are resting on the bottom of  the manifold channel. Check for correct positioning of sample cup arms. Check that the feet of the cups are not resting on a centering  protrusion in the channel.
	Incorrect strip placement.	Check that the strip is centered inside of the channel.
Sparking or burning in the  Immobiline® DryStrip gels	Current limit setting is too high.	Do not exceed the maximum recommended setting of 50 µA per Immobiline® DryStrip gel.
	Immobiline® DryStrip gel is not  fully rehydrated.	Ensure that the Immobiline® DryStrip gels are rehydrated with a sufficient volume of rehydration solution. Remove any large bubbles trapped under the Immobiline® DryStrip gel after placing it on rehydration solution.
	Immobiline® DryStrip gels dried out during IEF.	Always apply Immobiline® DryStrip Cover Fluid to prevent dehydration of rehydrated Immobiline® DryStrip gels.
Immobiline® DryStrips turn white and opaque after focusing	Immobiline® DryStrip gels dried out during IEF	Always apply recommended amount of Immobiline® DryStrip Cover Fluid to prevent dehydration of rehydrated Immobiline®  DryStrip gels.
Immobiline® DryStrip Cover Fluid overflows from Manifold	Excess cover fluid added.	Do not add more than the recommended volume. Ensure that the outside rim of the tray does not have any oil on it.