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Western blotting (WB) is regarded as one of the most sensitive techniques to detect specific proteins; the final step of a WB experiment is the visualization of specific proteins, with a labeled secondary antibody.
In western blot alkaline phosphatase (AP) and horseradish peroxidase (HRP) conjugated secondary antibodies are the most commonly use to detect your protein of interest in the middle of a complex protein mixture. While in immunohistochemistry (IHC) biotinylated secondary antibodies (learn more about why here) and HRP conjugated are used in IHC (get guide).
So using AP conjugated and HRP conjugated secondary antibodies optimized for western blot is important since it allows you to detect primary antibodies specific to your target protein raised in different species (e.g. goat, donkey and llama in the case of the VHH single domain).
HRP secondary antibodies
|Anti-Rabbit IgG VHH Single Domain Antibody (HRP) (ab191866)|
All products have been extensively tested and optimized for western blotting.
Cited within the scientific literature.
AP conjugated secondary antibodies can be routinely used at dilutions ranging from 1/5,000 to 1/50,000. Based upon a 1/5,000 dilution in 1500 blots with a single vial of an AP conjugated secondary.
HRP secondary antibodies can be used at dilutions ranging from 1/2,000 to 1/20,000. Assuming a 1/2000 dilution in 3ml milk/BSA, it is possible to perform 600 blots with a single vial of HRP-conjugated secondary.