A Powerful Technique for Large, Hard-to-Transfer Proteins - In-Gel Westerns

Tuesday, 20 October, 2015


  • in-gel western
  • westerns for large proteins
  • westerns for hard-to-transfer proteins
    • 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.

    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. 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 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.


    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.)

    Figure 2. Two-color In-Gel Western

    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 PBS-T alone, Intercept® 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 white paper on In-Gel Westerns: In-gel Immunochemical Detection of Proteins that Transfer Poorly to Membranes Michael J. Theisen and Mark L Chiu, Abbott Laboratories.

    For more information, refer to the In-Gel Western application pages.

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