Troubleshooting In-Gel Westerns – What can cause High Background?

So, you are doing an in-gel western because you have a difficult-to-transfer protein. Good for you!! But, you are seeing high background – and now you need some help to optimize your application.

What causes high background on In-Gel Westerns? Here are some possible causes with suggestions on how to solve or prevent the high background from occurring.

Stacking gel is still present.
– Cut the stacking gel away after electrophoresis.
Too much antibody.
– Reduce concentration of secondary antibody.
Uneven gel background may result from insufficient solution volumes for incubations.
– Use enough solution at each step (fixation, washes, and antibody incubations) to completely immerse the gel.
Pressing or squeezing gel during fixation and staining can cause splotchy background.
– Handle the gel gently, with gloved hands, and by the edges whenever possible.
Gel was not thoroughly washed.
– Use plenty of wash buffers to allow gel to move freely. Do not allow the gel to stick to bottom of container.


– Extend wash times or increase number of washes. Background may decrease if the gel is allowed to soak in PBS overnight at room temperature (protect from light).
Contaminated scanning surface.
– Before each use, apply methanol or ethanol followed by ultrapure water and wipe with lint-free tissues to remove residual dye. Remove any visible smears with isopropanol. Use canned air to remove any lint or dust.

Hopefully, after using some of these troubleshooting tips, you will get a nice gel image like this one:
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, MJ and Chiu, ML. In-gel immunochemical detection of proteins that transfer poorly to membranes. LI-COR Biosciences (2004).