Combining Acid Stripping With Photobleaching for Sequential Single-Channel Fluorescent Western Blot

Western blot (WB) is widely used for detecting specific proteins and their posttranslational modifications. The prevailing detection methods rely on either chemiluminescence or fluorescence. Fluorescence offers more stable signals, improves quantitative accuracy, and enables multiplex detection across various channels. Among these, the 800-nm near-infrared channel is preferred for its low background, superior signal-to-noise ratio, and compatibility with the inexpensive blocking agent, non-fat milk. In WB, it is often necessary to detect proteins with similar molecular weights or different forms of the same protein on the same blot. However, an optimal method for sequential probing using only the 800-nm channel has not been reported. Here, we demonstrate that combining 10% acetic acid (AA) stripping with photobleaching effectively eliminates 800-nm fluorophore signals from probed blots, enabling sequential probing within the same channel. The 10% AA solution disrupts antigen-antibody binding without causing protein loss from nitrocellulose membranes, achieving over 90% stripping efficiency for most tested antibodies and reducing potential steric hindrance during reprobing. Additionally, illumination with 800-nm light photobleaches residual signals. This approach provides a solution for sequential fluorescent WB using the optimal channel, delivering enhanced reliability and accuracy compared with chemiluminescent methods. Furthermore, this strategy can be extended to other imaging channels as needed.