January 1, 1970 (Vol. , No. )

Anton Posch, Ph.D.
Thomas Berkelman, Ph.D. Bio-Rad Laboratories

Analyze proteins better with these tips.

Two-dimensional gel electrophoresis (2DE) is a uniquely powerful technique that allows separation of up to thousands of proteins while retaining pI and MW information. Here is a list of 20 tips to help you get great 2DE results:

  1. Chemicals used for all steps in 2DE should be at least of analytical grade quality. Fresh solutions should always be made or stored as frozen aliquots at -70°C. Avoid repeated thawing and freezing of solutions and samples, and never reuse running buffer.
  2. Use clean and dust-free vessels to avoid keratin contamination.
  3. Keep the sample preparation strategy as simple as possible to avoid protein losses. When working with a new sample, use at least two different cell disruption protocols and compare protein yield (by protein assay) and qualitative protein content (by SDS-PAGE).
  4. Denaturation, solubilization, and disaggregation of proteins during sample preparation are time-dependent processes, so make sure to incubate proteins in 2DE lysis buffer for at least 1 hr at room temperature.
  5. Removal of interfering substances (salt, ionic detergents, nucleic acids, lipids, polysaccharides) is important for good results. Protease and phosphatase inhibitor cocktails should be added to the 2DE lysis buffer before cell lysis, if necessary.
  6. Insoluble material in the sample solution can clog the pores of the IPG strip. It is recommended to centrifuge the sample solution prior to IEF sample application for 1 h at 40,000 g.
  7. Do not heat urea-containing buffers above 37°C because protein carbamylation may occur.
  8. To yield proteins of interest at detectable levels, removal of interfering abundant proteins or nonrelevant classes of protein may be required. A simplified sample will yield better results.
  9. Knowing your protein concentration is very important. Be sure to quantitate your protein sample. About 5–10 µg/µL is a good concentration to aim for. Dilute the sample in rehydration solution to load the quantity best suited to the staining technique used and the experimental objective. Spot patterns are cleaner, sharper, and more reproducible when less protein is loaded into the IPG strip and a more sensitive stain is used.
  10. IPG strips have to be rehydrated to their original thickness of 0.5 mm. IPG strip rehydration should be performed for at least 12 h or overnight at 20°C. Cover the IPG strips with mineral oil during rehydration so they do not dehydrate.
  11. During IPG-strip rehydration, avoid trapping air bubbles between the IPG strip and the bottom of the rehydration tray. Make sure that the IPG strips don’t stick to the bottom of the rehydration tray. Both of these circumstances can result in incomplete rehydration and streaks in the gel.
  12. After rehydration, rinse and blot the IPG gel strips with deionized water to remove excess rehydration solution, otherwise urea crystallization on the surface of the IPG gel strips may occur.
  13. For better sample entry into the IPG strip, start IEF with a low-voltage gradient (200 V for 30–180 min) and limit current to 50 µA per IPG strip for the run. Focusing time depends on IPG strip length, pH gradient, gel additives, and the protein amount loaded. Recommended focusing temperature is 20°C.
  14. After completing the IEF run, IPG strips can be stored frozen at -70°C in rehydration trays or immediately applied to a second dimension SDS PAGE gel. Frozen IPG-strips can be stored for about 3–6 months.
  15. For the 2nd dimension gel, there should be no bubbles between the IPG strip and the gel well. Take care not to damage the well. Use warm, not hot, agarose to seal the IPG strip in place.
  16. Start at a low voltage to move the proteins from the IPG strip into the gel and then program the recommended voltage for the remainder of the run. Do not exceed the recommended voltage for the gel and running system. Excess voltage can lead to decreased resolution and distortions.
  17. Staining should be done at room temperature with gentle agitation. Ensure that the staining trays are dust free. Use a lid to keep out unwanted dust during staining. Handle gels by the edges and with gloves to prevent fingerprints and breakage.
  18. Since no stain is capable of staining all proteins, consider staining replicate gels with two or more different stains. Choose a stain that is appropriate for the protein load, imager, and downstream applications.
  19. Saturated images cannot be accurately quantitated. Reduce the imaging duration if images are saturated.
  20. Be sure to crop gel images used for analysis to the same size. Use the software warping option to get better spot matching results. Use the software normalization options to correct for loading variation between gels.

Two-dimensional gel electrophoresis can allow separation of thousands of proteins while retaining pI and MW information. [Bio-Rad]

Anton Posch, Ph.D., and Thomas Berkelman, Ph.D., are senior R&D scientists at Bio-Rad Laboratories. For a video tutorial to see how to run a 2DE experiment, please visit http://bit.ly/QEPpXF.

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