Signal detection

Chemiluminescent detection

It is based on visualization of the activity of HRP (Horse Radish Peroxidase enzyme), coupled to the secondary antibody, using chemiluminescent detection reagents like for example Agrisera ECL reagents:
AgriseraECL Bright (AS16 ECL-N) for low pico to mid-femtogram detection (for proteins of medium and high expression), or the enhanced AgriseraECL SuperBright (AS16 ECL-S) for extreme low femtogram detection (for proteins of low expression).

It is crucial to choose an ECL reagent of appropriate sensitivity. If the detection reagent applied is too sensitive, it will result with increased background signal, as shown here. Chemiluminescent signal is recorded  ith a CCD camera.

Chromogenic detection

For laboratories which do not have a CCD camera, visualization of the target protein can be made using so-called chromogenic detection using Agrisera BCIP/NBT ALP Substrate (AS19 BCIP-NBT) or Agrisera BCIP/NBT Plus ALP Substrate solution (AS19 BCIP-NBT-PLUS). In such a case, the secondary antibody is conjugated to an enzyme called ALP (Alkaline Phosphatase). The reaction is visualized directly on the membrane through color development, and the membrane can be stored in the lab book for a future reference. This detection system is not appropriate for proteins of low expression or to detect protein modifications, as phosphorylation.

Fluorescent detection

Fluorescent detection employs secondary antibody conjugated to a fluorescent dye, which will absorb light at a given wavelength and emit light of a higher wavelength, and does not involve use of any detection reagents. The fluorescent signal is recorded by a CCD camera. The advantage of this method is that it allows multiplexing more than one antibody per assay, and detection of normalization and loading controls on the same blot. Agrisera offers a wide range of secondary antibodies, conjugated to fluorescent dye, which can be used for signal visualization.

The complete collection of Agrisera's secondary antibodies can be found here.


Types of protein detection in Western blot

 Technical blog

Technical blog