Using Ampliflu™ Red Substrate for fluorescence-based Western Blotting

Ampliflu Red (Product No. 90101) is a fluorescence-based enzyme substrate for the specific and sensitive detection of HRP-labeled antibodies. It represents a viable alternative to the well-established chemiluminescent antibody-immunodetection procedure and has the advantage of a low background. Depending on the existing lab equipment, Ampliflu Red can be measured on a laser scanner with the corresponding settings or similar imaging systems. Standard protocols for the antibody-decoration-procedure can be used until the detection step. Incubation time is 5 minutes, as it is for most chemiluminescence-applications, but there is no signal accumulation necessary, only a simple fluorescence readout.

Ampliflu Red is converted by HRP-labeled secondary antibodies into highly fluorescent resorufin. Imaging is performed via excitation of the incubated membrane (e.g., using a laser-scanner) and employing an emission-filter (λex=571 nm / λem=585 nm). The limit of detection for the Ampliflu Red method is approximately 1 ng/band, and no extra time is necessary for signal accumulation as it is for chemiluminescence.

Schematic overview for the use of Ampliflu Red

Figure 1. Schematic overview for the using Ampliflu Red. The protein of interest is immobilized on a membrane after SDS-PAGE and Western blot-transfer, targeted by a primary antibody and followed by a secondary antibody carrying a horseradish-peroxidase (HRP) label. Ampliflu Red is converted into resorufin by the action of HRP. Imaging can by done by excitation of the resorufin (λex=571 nm / λem=585 nm) and fluorescence imaging.

Western Blot Detection

To receive an optimal signal-to-background ratio, use a low-fluorescence PVDF membrane for the Western blot transfer and block with 5% BSA. When working with Ampliflu Red, a standard protocol for the antibody decoration procedure can be used until the detection step. Incubation of the antibody-treated and washed membrane with a mixture of Ampliflu Red and H2O2 in PBS takes 5 minutes, and then the imaging can be done via laser excitation and an emission filter. No extra time for signal accumulation is necessary as it is for chemiluminescent signals. The use of Ampliflu Red is suitable for protein quantities between 1 ng and 1 µg per band of protein.

The example given in figure 2 shows the detection of FLAG-BAP protein after Western blot transfer on an Immobilon™-FL PVDF-Membrane by anti-FLAG antibodies, HRP-labelled secondary antibodies and the Ampliflu Red method. Imaging was performed on a FLA-3000 (Fuji) laser scanner with 532 nm excitation and a 580 nm emission filter, resulting in an image with low background. Other imaging systems are possible, with closely matching excitation sources and emission filter settings.

Detection of FLAG-BAP Protein

Figure 2. Western blot detection of FLAG-BAP using Ampliflu Red. FLAG-BAP protein (50 ng–1 ng) was separated on a 4–20 % SDS-PAGE, transferred to a low-fluorescence PVDF membrane by Western blot, blocked with 5 % BSA in PBS, incubated with Anti-FLAG® primary antibody, followed by rabbit-anti-mouse-IgG-HRP secondary antibody and visualized by Ampliflu Red. Imaging was done on a FLA-3000 (Fuji) laser scanner with 532 nm excitation and a 580 nm emission filter.

Ampliflu Red Protocol

Figure 3. Workflow for using Ampliflu Red after SDS-PAGE and Western blot-transfer. For membranes in the mini-format, 50 mL was used for the blocking and washing steps, and the antibody incubations were performed in 25 mL. The Ampliflu Red incubation mixture in PBS has a volume of 2 mL: 20 µL Ampliflu Red (create stock 10 mM in DMSO) and 100 µL H2O2 (create stock 20 mM in H2O). PBST contains 0.1% Tween-20 detergent.