Okay, so you're doing an in-gel western because you have a hard-to-transfer target (say, a glycoprotein) and because it gets rid of inconsistencies due to transfer. Plus, you are using near-infrared fluorescence detection for more consistent results (and, with an Odyssey® Imager, it is really fast and efficient to image and analyze).
Good for you!!
But, instead of seeing a beautiful gel like the one on the right, 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 was used.
- 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, or
- 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).
- The scanning surface was contaminated.
- 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, by reviewing these suggestions and working through your protocol, you WILL get an image just like the one above!
For more information on NIR In-Gel Westerns, visit our website.
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