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.