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In-Gel Westerns

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).

In-Gel Westerns: Detect protein directly in the polyacrylamide gel, without membrane transfer or blocking.

In-Gel Westerns are useful for:

  • Large, hydrophobic, or post-translationally-modified proteins, such as glycoproteins, receptors, or cell membrane proteins that do not transfer well
  • Small proteins that may pass through the membrane during transfer
  • Benefits of In-Gel Western detection:

  • Two-color detection of two different protein targets can be performed in the gel when using infrared fluorescent secondary antibodies and near-infrared imaging systems
  • Saves time, reduces cost, and eliminates variables associated with membrane transfer and blocking
  • Offers sensitivity comparable to chemiluminescent in-gel detection, but not as high as membrane detection
  • To perform an infrared fluorescent In-Gel Western, refer to In-Gel Western Protocol.

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