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)

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.

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).
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).

Optimizing your Near-infrared Fluorescent In-Gel Westerns

In-Gel Western with Two-Color Detection A Powerful Technique for Large, Hard-to-transfer Proteins
The In-Gel Western detection protocol may require optimization for each target protein or gel type. Sensitivity of In-Gel Westerns may be lower than standard Western blots. (Transfer to a membrane concentrates the target protein, whereas in gels, protein is dispersed through the thickness of the gel.)

Use the following guidelines for optimization:

  • Optimization of primary and secondary antibody dilutions, as well as amounts of Tween® 20 in diluted antibodies, may be needed to achieve maximum signal and minimum background. Recommended Tween 20 concentration is 0.1%.
  • Try different buffers for dilution of the antibodies, including PBST alone, Odyssey Blocking Buffer, or milk. Changing the buffer solution may dramatically improve performance.
  • To avoid background issues, use high-quality ultrapure water. Rinsing previously used incubation boxes or trays with methanol can reduce background contamination on gels.
  • For experiments utilizing streptavidin labeled with IRDye® infrared dyes, add 0.01% SDS in addition to Tween 20 in the antibody diluents and wash buffer.

Here is a nice white paper reference on In-Gel Westerns:
In-gel Immunochemical Detection of Proteins that Transfer Poorly to Membranes
Michael J. Theisen and Mark L Chiu, Abbott Laboratories

Detect Difficult Proteins More Easily with Near-Infrared In-Gel Westerns

Western blot detection of proteins requires separation of protein mixtures by electrophoresis, followed by transfer of the separated proteins to nitrocellulose or PVDF membranes for detection. In-Gel Western detection avoids transfer problems by directly detecting target proteins within the polyacrylamide gel matrix, using the Odyssey® CLx or Classic Infrared Imaging System or the Odyssey Fc Imaging System.

The Odyssey Infrared Imaging systems allow you to detect target proteins while still embedded in the gel – without transfer to a membrane – using near-infrared secondary antibodies, such as the LI-COR® IRDye Conjugates. Using near-infrared fluorescence detection methods for In-Gel Westerns makes this a powerful technique. It saves time, reduces cost, and eliminates the variables introduced by the transfer step or subsequent blocking of the membrane. In-Gel Western detection can be performed with standard Odyssey reagents – no special kit is required.

Comparing Odyssey Infrared Detection of In-Gel Westerns vs. Chemiluminescence Detection

Figure 1. Sensitivity of Odyssey infrared In-Gel Westerns is equal to or better than chemiluminescence. Beginning with 10 ng/lane (far left), two-fold serial dilutions of purified Transferrin were separated by electrophoresis on duplicate gels. In-Gel Westerns were detected with infrared fluorescence (top) and chemiluminescence on film (bottom). Odyssey detection outperformed chemiluminescence.

 

For more information, refer to Odyssey® In-Gel Western Detection Protocol and the In-Gel Western application pages.