Minicelda ZOOM™ IPGRunner™

1) Trim excess plastic from IPG strip

2) Equilibrate IPG strip for 2 x 15 min in 5 mL of the appropriate buffer (Use NuPAGE sample buffer for NuPAGE ZOOM gels and Tris Glycine ZOOM gels). Equilibration buffer: 1X NuPAGE sample buffer, 50 mM DTT.

3) Place equilibrated IPG strip in large well of ZOOM gel. Seal the strip in place with an agarose (0.5%) overlay.

4) Load molecular weight markers in small well of ZOOM gel.

Variations: To alkylate the proteins after reducing them, prior to separation on ZOOM gels: 1) Incubate IPG strip for 15 min in equilibration buffer 2) Transfer strip to Equilibration Buffer containing 125 mM iodoacetamide and lacking reducing agent. Incubate an additional 15 min. To denature the proteins with urea after IEF: 6 M urea can be added to Equilibration Buffer, if desired.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Here are the possible causes and solutions:

- Low protein load. Increase the protein load. Use an accurate and sensitive protein estimation method.
- Improper sample preparation. Increase solubilization reagents. Use at least 8 M urea for solubilization. Add DTT and non-ionic detergents (see manual for details)
- Strip not correctly oriented. Align the strip correctly as described in the manual. Be sure to have the gel side up when loading the strip into the ZOOM IPGRunner Cassette.
- Air bubbles between the strip and 2D gel. Smooth out any air bubbles.
- Insensitive detection method. Use sensitive detection methods such as silver staining or immunoblotting.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

A possible reason is poor contact between electrodes or incomplete circuit. Make sure that you have added 600 µL deionized water to the Electrode Wicks and the gel is exposed at the anodic and cathodic ends of the cassette. Check the power supply. Be sure to set the “Load Check” to off to enable the power supply to operate at low current.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

The ZOOM IPGRunner System should be used with an external DC power supply designed for electrophoresis applications. This power supply must:

- Be isolated from the ground so that the DC output is floating (safety requirement).
- Be programmable, with a 4-protocol minimum.
- Be able to operate at low current (less than 1 mA) as IEF is performed at very low current
Note: Many power supplies automatically shut off when the current drops below 1 mA. You will need a power supply capable of overriding the low current shut-off feature. The electrical leads of the ZOOM IPGRunner Lid are recessed and may not fit into some power supply units. To allow connection of the ZOOM IPGRunner power leads with certain power supplies, use Invitrogen Power Supply Adapters available separately (Cat. No. ZA10001).

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

The ZOOM IPGRunner Mini-Cell is impervious to alcohol, but not compatible with chlorinated hydrocarbons (e.g., chloroform), aromatic hydrocarbons (e.g., toluene, benzene) or acetone.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.