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