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Two-Color In-Gel Western Detection

Multiplex detection of two target proteins by In-Gel Western.

Figure 1. Multiplex detection of two target proteins by In-Gel Western. Two-fold dilutions of a T7-Tag extract (red; 700 nm) and purified Transferrin (green; 800 nm) were detected using the In-Gel Western protocol and IRDye® secondary antibodies. Gel was imaged with the Odyssey® CLx Imager. Reprinted from Urh, M. et al. Poster presentation. Advances in Genome Biology and Technology Conference (2002).

Primary and secondary antibodies must be carefully selected for two-color detection or cross-reactivity will result. Follow these guidelines for two-color detection:

  • All secondary antibodies must be highly cross-adsorbed to eliminate cross-reactivity.
  • The two primary antibodies used must be derived from different host species so they can be discriminated by secondary antibodies of different specificities.

    Example: rabbit anti-protein X + mouse anti-protein Y primary antibodies

  • The two secondary antibodies used must be derived from the same host species so they will not react against one another. The secondary antibodies should not recognize immunoglobulins from other species that may be present in the sample.

    Example: goat anti-rabbit IgG + goat anti-mouse IgG

  • One secondary antibody should be labeled with IRDye® 800CW (800 nm channel) and the other with IRDye 680LT or IRDye 680RD (700 nm channel), or other commercially available near-infrared dyes.
  • Always perform preliminary blots with each antibody alone to determine the expected banding pattern for each, before combining them in a two-color experiment. Slight cross-reactivity may occur, particularly if the antigen is very abundant, and can complicate interpretation of your blot. If cross-reactivity is a problem, load less protein or reduce the amount of antibody.
  • For best results, avoid using primary antibodies from mouse and rat together for a two-color experiment. Because the species are so closely related, it is not possible to completely adsorb away cross-reactivity. Substantial cross-reactivity between bands may occur. If using mouse and rat together, it is crucial to run single-color blots first with each individual antibody to be certain of expected band sizes.

Protocol Modifications for Two-Color Detection

For two-color detection, follow Sample Protocol: One-color Detection with the following modifications:

  • Use two labeled secondary antibodies that are labeled with dyes that are detected in two different channels. Example: IRDye 680LT or IRDye 680RD (700 nm) and IRDye 800CW (800 nm)
  • Make sure that antibody specificities and hosts are appropriate and will not cross-react.
  • Combine the two primary antibodies in antibody diluent in step 4, and incubate simultaneously with the gel.
  • Combine the two IRDye secondary antibodies in the antibody diluent in step 6. Incubate simultaneously with the gel.


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