Okay, 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!