Ampliflu Red

Sigma-Aldrich® now offers a fluorescence-based enzyme substrate, Ampliflu Red (Product No. 90101). It is a reliable new tool 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 very 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 it comes to the detection step. Incubation time is 5 min, 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 about 1 ng/band, and no extra time is necessary for signal accumulation as it is for chemiluminescence.

Figure 1. Schematic overview for the use of 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.

Westernblot Detection
In order to receive an optimal signal to background ratio, we strongly recommend to 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 it comes to the detection step. Incubation of the antibody-treated and washed membrane with a mixture of Ampliflu Red and H2O2 in PBS takes 5 min, 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.

Figure 2. 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 (1:1000), followed by rabbit-anti-mouse-IgG-HRP secondary antibody (1:10000) 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.

Figure 3. Protocol for the use of Ampliflu Red after SDS-PAGE and western blot-transfer. For membranes in the mini-format, the blocking and washing steps were done in a volume of 50 ml, whereas the antibody-incubations were done 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.

back to top