Troubleshooting Near-Infrared In-Gel Westerns - No Signal

Tuesday, 17 November, 2015


  • in-gel westerns
  • large protein western
  • glycoproteins and westerns

    • You have decided to try an In-Gel Western because you read that they are sueful for large, hydrophobix, or post-translationally-modified proteins. Your protein is a large cell membrane protein, so you thought this technique would be perfect.

    But, what you are seeing, or, um, well, NOT seeing is a signal. Rats! Where IS it? Where is your protein?

    Well, here are some possible causes with ways you can troubleshoot this 'no signal' issue:

    • Not enough antibody.
      • Increase amount of primary and/or secondary antibody. Extend primary antibody incubation to overnight at 4°C to increase signal.
      • Remember that In-Gel detection is not as sensitive as blot detection; adjust sample loading and antibody concentrations accordingly.
    • Antibody dilution buffer is not optimal for your primary antibody.
      • Try a different dilution buffer; this can significantly affect performance of some primary antibodies.
      • Suggested buffers include 3-5% BSA, Intercept® (PBS) Blocking Buffer, or Intercept (TBS) Blocking Buffer, and PBS or TBS (all with 0.1% Tween® 20). Other blockers (milk, casein, commercial blockers) and Tween 20 concentrations can also be tested.
    • Gel type is not optimal.
      • Amresco NEXT gels or NuPAGE® Bis-Tris pre-cast gels are recommended for In-Gel detection. Other commercial gel sources and homemade gels can be used, but may show reduced sensitivity and require further optimization.
    • Antibody did not penetrate gel sufficiently or evenly.
      • Acrylamide percentage was too high. Try a lower percentage or a gradient gel.
      • Increase volume for antibody incubations so that gel is completely immersed in antibody solution.
      • Make sure gel is adequately fixed. Some monoclonal antibodies may be sensitive to residual acid in the gel; in this situation, eliminate acetic acid from the fix or extend the water wash step.
    • Gel was left in isopropanol/acetic acid too long.
      • This may cause protein to be lost from the gel. Fix for 15 minutes only.

    Whew! Well, hopefully by using one of these tips, you are NOW seeing a signal from your protein. For more information on NIR In-Gel Westerns, visit our website.

    Figure 3. In-Gel detection of Cytochrome P450 3A4 (CYP3A4). Fixed gel was probed with anti-CYP3A4 primary antibody and IRDye® 800 secondary antibody. The limit of detection is approximately 3 ng. Reprinted with permission from Theisen, M. J. and Chiu, M. L. LI-COR Biosciences (2004).

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