The second antibody plays an important role in immunology experiment, and the selection of the second antibody also determines the success or failure of the whole experiment.
The following points must be paid attention to when selecting the secondary antibody in the experiment:
(1) The type of the secondary antibody is determined mainly according to the origin of the species of the primary antibody. If the primary antibody is from mice, the secondary antibody can be purchased from mice (such as goat anti mouse, rabbit anti mouse, etc.).
(2) Selection of markers. Generally speaking, probes coupled to the secondary antibody mainly include enzymes (horseradish peroxidase HRP and alkaline phosphatase AP or their derivatives APAAP, PAP), fluorescent groups (FITC, RRX, TR, PE) and biotin. The choice of the second antibody of the probe mainly depends on the specific experiment. For Western blot and ELISA, the most commonly used secondary antibody is enzyme labeled secondary antibody, while fluorescent group labeled secondary antibody is usually used in cell or tissue labeling experiments (cellular immunochemistry, histochemistry, flow cytometry), and horseradish peroxidase or alkaline phosphatase labeled secondary antibody can also be used in immunohistochemistry. If you want to amplify the detection signal to a greater extent, you can use the biotin/avidin detection system.