Troubleshooting In-Gel Westerns – Where’s the Signal?

In-Gel Western with Two-Color DetectionOkay, so you’re doing an in-gel western because you have a hard-to-transfer target (say, a glycoprotein). And you are using near-infrared fluorescence detection because it gets rid of inconsistencies due to transfer (and with an Odyssey it’s really fast and efficient to image and analyze!)

You read the In-Gel Western troubleshooting blog from March 6, 2012, but right now, what you’re seeing, or, um, well, NOT seeing is a signal. Rats! Where IS it?

Well, here are some possible causes with ways to solve or prevent this from happening:

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, Odyssey Blocking Buffer (PBS), or Odyssey Blocking Buffer (TBS), 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. Stay tuned for more troubleshooting tips for near-infrared fluorescent In-Gel Westerns in future blogs!
In-Gel detection of Cytochrome P450 3A4 (CYP3A4).

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)