The In-Gel detection protocol may require optimization for each target protein or gel type. Sensitivity of In-Gel Westerns is typically lower than for 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 PBS-T 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.